Methods for monitoring the status of assays and immunoassays

ABSTRACT

The invention relates in part to the use of independent assay controls (IACs) for the optical communication between an assay device and an instrument in monitoring and performing assays, preferably immunoassays.

RELATED APPLICATIONS

The present application is related to U.S. patent application Ser. No.09/003,090 entitled “Immunoassay Fluorometer,” Buechler et al., filedJan. 5, 1998, now U.S. Pat. No. 6,830,731, and U.S. patent applicationSer. No. 09/003,066 entitled “Media Carrier for an Assay Device,”Buechler et al., filed Jan. 5, 1998, now U.S. Pat. No. 6,074,616, eachof which are filed concurrently herewith and each of which areincorporated herein by reference in their entirety, including allfigures, tables, and drawings.

FIELD OF THE INVENTION

This invention relates in part to the use of independent assay controls(IACs) for the optical communication between an assay device and aninstrument in monitoring and performing assays, preferably immunoassays.

BACKGROUND OF THE INVENTION

The development of reliable methods for rapidly and simply measuringanalytes in complex samples has become increasingly important. Forexample, the point of care testing in hospital emergency departmentsrequires unskilled technicians to perform complex chemical andimmunochemical assays to rapidly define the status of patients. Thetesting is usually performed by a nurse or an emergency room technicianwho are not trained as clinical chemists. The current practice ofsending blood samples to the hospital laboratory is not feasible whenthe results are required within 30 min. The problem is thus that assayresults are needed in a rapid time but the testing protocols, personneland equipment available to the hospital emergency department are notcompatible with this need. Other scenarios for obtaining rapid resultsthrough simple methods are in physicians offices, in patient homes andin field testing of pollutants and contaminants.

There is thus an unmet need for an immunoassay system that is simple,rapid and reliable.

Reliability in an immunoassay system is critical for the accuratemeasurement of the analyte. In an emergency room setting, the assayresults can guide the physician in diagnosing and treating the patient.In a home setting, the assay result can, for example, help determine theamount and frequency of a therapeutic drug. In the field testing ofpollutants and contaminants, the testing can define the extent ofrenovation or ground excavation needed to remove the contaminant.

Previous references regarding assay controls have not clearly definedparameters that require evaluation in assay devices. For instance, manypublications simply provide examples of controls that determine theeffect of non-specific binding. Some of these publications relate mainlyto methods of controlling for non-specific binding and some referencesrelate to devices that incorporate controls for non-specific binding.See, e.g., U.S. Pat. Nos. 4,533,629, 4,540,659, 4,843,000, 4,849,338,5,342,759, 4,649,121, 4,558,013, 4,541,987, 4,472,353, and 4,099,886.

SUMMARY OF THE INVENTION

The disclosure provided herein teaches the novel use of independentassay controls (IACs) in assay devices. IAC results are not typicallydependent upon assay results, however, results from one IAC may bedependent upon results from another IAC. For example, a change in ameasurement in one IAC may be proportional to a change in a measurementin another IAC if the two IACs are dependent upon one another.

The term “IAC” as used herein can refer to any assay control measurementthat is independent of an assay measurement. Some IACs may beindependent with respect to one another and other IACs may be dependentwith respect to one another. For example, a change in a first IACmeasurement may occur while a change in a second IAC measurement may notoccur when IACs are independent. In another example, a change in a firstIAC measurement may correlate with a proportionate change in a third IACmeasurement when IACs are dependent with respect to one another.

Once one or more IACs are measured, the IAC measurements can be utilizedto correct assay measurements. These IACs can ensure that the resultsobtained from an assay detection system are accurate when assayconditions vary. Any of the IACs of the invention can be utilized tocorrect assay measurements. One IAC may be utilized to correct assayresults, or multiple IACs may be utilized to correct assay results.

IAC for Determining the Rate of Flow

The term “rate of flow” as used herein can refer to the velocity atwhich a liquid solution travels through an assay apparatus. Rate of flowcan be measured in terms of distance per unit time. Alternatively, rateof flow can be expressed in terms of an arbitrary unit or as a deviationfrom a mean value for rate of flow. A mean value for rate of flow can bedetermined in multiple experiments using different assay devices.

The term “assay device” as used herein can refer to any appropriateconstruction that allows the flow of fluids through chambers. Forexample, at least some chambers in an assay device may be tubes that candraw fluid by capillary action. Assay devices can be constructed fromnearly any type of material, including propylene, polypropylene, andplastics, for example. An assay device may be placed in an apparatusdescribed herein. The invention relates in part to any assay devicecapable of carrying out an IAC method defined herein. For example, if anIAC method requires that a second member of a binding pair (MBP) isassociated with a solid phase of a diagnostic lane in an assay device,then one aspect of the invention features an assay device that comprisesa second MBP associated with a solid phase in a diagnostic lane.

Thus in a first aspect, the invention features a method for determininga rate of flow of a solution through an assay device. The assay devicecomprises a reaction chamber and at least one diagnostic lane. Themethod comprises the following steps: Step (a): providing a first memberof a binding pair (MBP) in the reaction chamber and a second MBP boundto a solid phase in the diagnostic lane. The first MBP comprises alabel, and the first MBP and said second MBP do not appreciably bind toany IAC assay reagents in the assay device. However, the first MBP andthe second MBP have specific binding affinity for one another. Step (b):detecting a signal in the diagnostic lane, where the signal is generatedfrom the label. Step (c): determining the rate of flow of liquid throughthe assay device from the reaction chamber through the diagnostic lanefrom the amount of the signal in the diagnostic lane.

The term “reaction chamber” as used herein can refer to a portion of anassay device that contacts fluid before fluid reaches a diagnostic laneor diagnostic zone. A reaction chamber can be coextensively formed withan assay device, or alternatively, a reaction chamber can be a separatecomponent with respect to an assay device. For example, samples andexogenously added reagents can be mixed in a test tube, which can serveas a reaction chamber. A portion or the entire contents of this testtube can then be introduced to an assay device. Fluids, biologicalsamples, and reagents may be directly added to a reaction chamber.

The term “diagnostic lane” as used herein can refer to a portion of anassay device that harbors components comprising an IAC and an assay. Adiagnostic lane may be as simple as a region allowing opticalmeasurement of a signal. Alternatively, a diagnostic lane may comprisecomponents that have specific binding affinity for molecules thatcomprise labels and molecules that comprise labels as a result of theassay process, such that binding events can be detected. In an exampleof an apparatus that includes capillary tubes, diagnostic lanes canembody capillary tubes aligned in parallel with respect to onedimension, or capillary tubes aligned in series with respect to onedimension.

The term “member of a binding pair (MBP)” as used herein can refer toany molecule or conglomerate of molecules that forms a complex withanother molecule or conglomerate of molecules through specific bindingevents. Examples of MBPs are antibodies and their corresponding MBPs, aswell as receptors and their corresponding MBPs. MBPs may be comprised ofproteins, polypeptides, and/or small molecules, for example.

The term “do not appreciably bind” as used herein can refer to aphenomenon where interactions and/or lack of interactions between MBPsand other assay reagents do not significantly interfere with detectionof a signal.

The term “assay reagents” as used herein can refer to any moleculeslocated in an assay device or molecules exogenously added to a fluidintroduced to an assay device used for measuring the presence or amountof an analyte. For example, one or more assay reagents may beincorporated into an assay device at a specific location of the assaydevice during the manufacture of the assay device. Such an assay reagentmay serve a function of forming a complex with one or more components ina biological fluid to provide an assay result. In another example, anassay reagent may be exogenously added to a biological fluid to form acomplex with one or more molecules in the fluid. Examples of analytesare cyclosporin hCG, CKMG, troponin and myoglobin.

The term “providing” or “provided” as used herein can refer to placingan assay reagent, test sample, or any other type of mixture of moleculesinto an assay device. An assay reagent can be provided to a reactionchamber of an assay device by delivering a solution comprising the assayreagent into the reaction chamber via a pipet, for example. In anotherexample, an assay reagent or any other type of molecules may be providedin a diagnostic lane of an assay device by linking the molecules to asolid phase of the diagnostic lane. Methods for linking molecules to asolid support are well known to a person of ordinary skill in the artand are described further herein.

The term “test sample” as used herein can refer to any solution placedin an assay device. The solution can be extracted from a biologicalorganism or specimen, for example. In another example, a test sample maybe created in vitro and then be placed in an assay device. A test samplemay also exist as a combination of these aforementioned examples.

The term “specific binding affinity” as used herein can refer to aphenomenon where a first molecule can form a complex with a secondmolecule with a higher probability as compared to a complex formedbetween the first molecule and a third molecule. In this example, thefirst molecule has specific binding affinity for the second molecule.

The term “label” as used herein can refer to any molecule that may belinked directly or indirectly to an MBP or reagent. A label may belinked to an MBP or reagent by covalent bonds or attractive forces suchas hydrophobic, ionic, and hydrogen bond forces. A label may emit asignal, which is described hereafter.

The term “signal” as used herein can refer to any detectable parameter.Examples of these parameters include optical, electrical, or magneticparameter current, fluorescent emissions, infrared emissions,chemiluminescent emissions, ultraviolet emissions, light emissions, andabsorbance of any of the foregoing. A signal, for instance, may beexpressed in terms of intensity versus distance along a diagnostic laneof an assay device. In addition, a signal may expressed in terms ofintensity versus time. The term “signal” can also refer to the lack of adetectable physical parameter. The invention teaches methods, apparatus,and kits that can simultaneously measure multiple signals and multipletypes of signals.

The term “amount” as used herein can refer to an increase, decrease,and/or maintenance of the intensity of physical parameter within aspecific level of sensitivity. An amount of a signal can be expressed interms of arbitrary units. For example, an increase in the amount of asignal can be detected if a signal increases by ten units and thesensitivity for measuring the signal is within one unit.

In preferred embodiments, the first MBP, second MBP, and other reagentsare selected from the group consisting of binding protein, antibody,antibody fragment, protein, peptide, and organic molecule.

The term “binding protein” as used herein can refer to any protein thathas specific binding affinity for one or more molecules. A bindingprotein can embody a protein extracted from a cell or may embody aportion of such a protein, for example. A binding protein may also besynthesized in vitro using methods well known to a person of ordinaryskill in the art. A binding protein may also be a molecule thatcomprises multiple binding regions and/or multiple binding proteins.Binding proteins can have specific binding affinity to proteins,polypeptides, and organic molecules, for example. Examples of bindingproteins are membrane receptors, non-membrane receptors, and antibodies.

The term “antibody” as used herein can refer to any molecule havingamino acid similarity and/or structural similarity to the immunoglobulinclass of biological molecules. An antibody may exist as animmunoglobulin extracted from a patient or animal. Alternatively, anantibody may exist as a portion of an immunoglobulin. One of theseportions can be referred to as an “antibody fragment.” Preferably, anantibody fragment harbors all of the hypervariable region of animmunoglobulin or a portion of the hypervariable region of animmunoglobulin. Antibodies can be extracted from properly immunizedanimals and can be synthesized by recombinant expression techniques inbacteria, for example.

The term “organic molecule” as used herein can refer to any moleculethat comprises a covalent bond between carbon, nitrogen, oxygen, and/orsulfur. An organic molecule can range in size from carbon monoxide tolarge complex polymers. Organic molecules can relate to glycosidemolecules. Examples of antibodies and binding proteins having specificbinding affinity for organic molecules are well known in the art.

Any IAC and assay reagents and/or MBPs can be dissolved or exist insuspension in fluids and fluid analogs. Alternatively, IAC and assayreagents and/or MBPs can be linked to a solid support or a solid phase.

The term “solid support” and the term “solid phase” as used herein canrefer to a non-liquid substance. A solid support may be coextensivelyformed with an assay device (e.g., a solid support can be a membrane ora portion of a capillary tube in an assay device), or alternatively, asolid support may be small diameter beads that flow through an assaydevice. Reagents may be associated with a solid support or a solid phaseby covalent bonds and/or via non-covalent attractive forces such ashydrogen bond interactions, hydrophobic attractive forces, and ionicforces, for example.

In preferred embodiments, the label is selected from the group ofmolecules consisting of dye, fluorescence emitting dye, achemiluminescence emitting dye, infrared emitting dye, colloidal sol,molecule that generates an electrical and/or magnetic signal, andenzyme.

The term “dye” as used herein can refer to any molecule that detectablyemits or absorbs visible light, ultraviolet light, infrared light,fluorescently-derived light, chemiluminescently-derived light, and/orany other type of optically detectable parameter.

The term “colloidal sol” as used herein can refer to a partiallydispersed conglomerate of molecules existing as a solid, gel, or liquid.

In another aspect, the invention features an apparatus for determining arate of flow of a solution. An apparatus can comprise: (a) an assaydevice comprising a reaction chamber and at least one diagnostic lane;(b) an optical component; and (c) a signal processor. The rate of flowcan be determined by providing a first member of a binding pair (MBP) inthe reaction chamber, and a second MBP bound to a solid phase in thediagnostic lane. The first MBP comprises a label. Although the first MBPand the second MBP do not appreciably bind to any assay reagents in theassay device, the first MBP and the second MBP have specific bindingaffinity for one another. The optical component can detect a signal,where the signal is generated from the label. The signal processor candetermine the rate of flow of liquid through the assay device from thereaction chamber through the diagnostic lane from the amount of thesignal in the diagnostic lane.

The term “optical component” as used herein can refer to any componentof an apparatus through which an assay signal passes. For example, anoptical component may in part convert a fluorescent signal into anelectrical signal by allowing the fluorescent signal to pass through itand into a signal processor. An optical component may be synthesizedfrom glass, plastic, or crystalline materials. An optical component mayoperate in conjunction with other devices in an apparatus to convert anassay signal into another signal. An optical component may also exist asan independent device that is not fused to an apparatus of theinvention, or may be coextensively formed with an assay device of theinvention.

The term “signal processor” as used herein can refer to a component ofan apparatus that in part modifies an assay signal. For example, asignal processor may operate in conjunction with an optical component ofan apparatus to convert a fluorescent signal into an electrical signal.In another example, a signal processor may smooth an assay signal, asdescribed hereafter. In yet another example, a signal processor maycorrect an assay measurement by utilizing one or more IAC measurementsin an assay device. Signal processors may also be referred to asco-processors for the purposes of this invention. Processors andco-processors can operate in conjunction with a media carrier, describedhereafter.

In yet another aspect, the invention features a kit for determining arate of flow of a solution. A kit can comprise: (a) at least one of aFood and Drug Administration label and a set of instructions; and (b) anapparatus comprising: (i) an assay device comprising a reaction chamberand at least one diagnostic lane; (ii) an optical component; and (iii) asignal processor. The rate of flow can be determined by providing afirst member of a binding pair (MBP) in the reaction chamber, and asecond MBP bound to a solid phase in the diagnostic lane. The first MBPcomprises a label. Although the first MBP and the second MBP do notappreciably bind to any assay reagents in the assay device, the firstMBP and the second MBP have specific binding affinity for one another.The optical component can detect a signal, where the signal is generatedfrom the label. The signal processor can determine the rate of flow ofliquid through the assay device from the reaction chamber through thediagnostic lane from the amount of the signal in the diagnostic lane.

The term “Food and Drug Administration label” as used herein can referto any label that has been approved by a Food and Drug Administration inthe United States or any other similar administration in anothercountry. In addition, the term may refer to a label that indicates thata certain compound or apparatus has been approved by a Food and DrugAdministration in the United States or any other similar administrationin another country.

The term “set of instructions” as used herein can refer to text and/ordiagrams that can aid an operator of a method, apparatus, and/or kit ofthe invention. For example, a set of instructions may take the form of astep wise set of instructions or may exist as text on the outside of abox that contains an apparatus and/or assay device of the invention.

IAC for Determining Environmental Conditions

The term “environmental conditions” as used herein can refer to physicalparameters within an assay device. For example, an IAC may determine theeffect of diffusive properties of reagents that can govern the rate atwhich a molecule or molecules dissolve into a fluid and/or an oncomingfluid front. The proximity of IAC reagents in a reaction chamber and inan assay device, in general can define functions related to non-specificbinding of label, flow mechanics, incubation time, homogeneity of thereaction mixture, and sample matrix including hematocrit of blood andthe degree of heterophilic antibodies in blood of the assay. IACs of theinvention can be utilized to correct assay measurements upon changingphysical attributes of an assay device and/or fluids introduced to assaydevices.

Hence, in another aspect, the invention features a method fordetermining environmental conditions in an assay device during an assay,where the assay device comprises a reaction chamber and at least onediagnostic lane. The reaction chamber comprises a first MBP and a secondMBP arranged to form a solution with a test sample, where the first MBPcomprises a label, and where the second MBP comprises an affinity tag.The diagnostic lane comprises an affinity tag partner (ATP), and the ATPhas specific binding affinity to the affinity tag. The first MBP, thesecond MBP, the ATP, and the affinity tag do not appreciably bind to anyIAC or assay reagents in the assay device. The first MBP and the secondMBP have specific binding affinity for one another. The method comprisesthe steps of: (a) detecting a signal in the diagnostic lane, where thesignal is generated from the label, and where the signal is detected ata location in a position where the ATP is located; and (b) determiningthe environmental conditions in the assay device during assay, where theenvironmental conditions are related to the amount of the signal in thediagnostic lane.

The term “affinity tag” as used herein can refer to a molecule linked toa reagent and/or MBP, which has specific binding affinity to an affinitytag partner. An affinity tag can be linked to a reagent and/or MBP by acovalent bond or by attractive forces described previously. An affinitytag can be linked to a reagent or MBP after the reagent or MBP issynthesized, or alternatively, an affinity tag may be linked to areagent or MBP while the reagent or MBP is being synthesized. Anaffinity tag may be an organic molecule, a protein, a glycosyl moiety,or a polypeptide, for example.

The term “affinity tag partner” as used herein can refer to any moleculethat has specific binding affinity to an affinity tag. An affinity tagpartner may be associated with a solid phase or may exist as anunattached molecule that can freely diffuse in solution. An affinity tagpartner may be an organic molecule, a polypeptide, a glycosyl moiety, ora protein, for example. Examples of affinity tag/affinity tag partnercomplexes that are well known in the art are hemagglutininpeptide/hemagglutinin peptide antibody complexes and avidin/biotincomplexes.

In a preferred embodiment, the first MBP and the second MBP areassociated with at least one of a lid and a base of the reactionchamber. In other preferred embodiments, the ATP is associated with asolid support in the diagnostic lane.

In another aspect, the method comprises the step of introducing thesecond MBP and the ATP to the reaction chamber, where the ATP comprisesa second affinity tag, and where the diagnostic lane comprises a secondATP. The second ATP has specific binding affinity for the secondaffinity tag, and the second ATP and the second affinity tag do notappreciably bind to the assay reagents.

In a preferred embodiment, the first MBP, second MBP, and reagents areselected from the group consisting of binding protein, antibody,antibody fragment, protein, peptide, and organic molecule. In anotherembodiment, the label is selected from the group of molecules consistingof dye, fluorescence emitting dye, chemiluminescence emitting dye,infrared emitting dye, colloidal sol, molecule that generates anelectrical and/or magnetic signal, and enzyme.

In another aspect, the invention features an apparatus for determiningenvironmental conditions in an assay device during an assay. The kit cancomprise: (a) the assay device; where the assay device comprises areaction chamber and at least one diagnostic lane; (b) an opticalcomponent; and (c) a signal processor. The reaction chamber can comprisea first MBP and a second MBP arranged to form a solution with a testsample. The first MBP may comprise a label and the second MBP maycomprise an affinity tag. The diagnostic lane can comprise an affinitytag partner (ATP) and the ATP may have specific binding affinity to theaffinity tag. Although the first MBP, the second MBP, the ATP, and theaffinity tag do not appreciably bind to any assay reagents in the assaydevice, the first MBP and the second MBP have specific binding affinityfor one another. The optical component may detect a signal, where thesignal is generated from the label. The signal processor can determinethe environmental conditions in the assay device during assay, where theenvironmental conditions are related to an amount of the signal in thediagnostic lane.

In yet another aspect, the invention features a kit for determiningenvironmental conditions in an assay device during an assay. A kit cancomprise: (a) at least one of a Food and Drug Administration label and aset of instructions; (b) an apparatus, comprising: (i) the assay device;where the assay device comprises a reaction chamber and at least onediagnostic lane; (ii) an optical component; and (iii) a signalprocessor. The reaction chamber can comprise a first MBP and a secondMBP arranged to form a solution with a test sample. The first MBP maycomprise a label and the second MBP may comprise an affinity tag. Thediagnostic lane can comprise an affinity tag partner (ATP) and the ATPmay have specific binding affinity to the affinity tag. Although thefirst MBP, the second MBP, the ATP, and the affinity tag do notappreciably bind to any assay reagents in the assay device, the firstMBP and the second MBP have specific binding affinity for one another.The optical component may detect a signal, where the signal is generatedfrom the label. The signal processor can determine the environmentalconditions in the assay device during assay, where the environmentalconditions are related to an amount of the signal in the diagnosticlane.

IAC for Determining Assay Progress and Time of Completion

The term “progress” and “time of completion” as used herein can refer tomonitoring the flow of molecules through an assay device. For example, atime of completion can be determined from a measure of signal intensitywith respect to time and/or distance in a diagnostic lane. The rate ofchange of the amount of the signal at any point in the read-out or theabsolute intensity of the signal at any point in the read-out candetermine the progress and time of completion of the assay. Theseparameters are discussed in more detail hereafter.

Thus, in another aspect, the invention features a method for measuringthe progress and time of completion for an assay in an assay device,where the assay device comprises a reaction chamber and at least onediagnostic lane. The method comprises the steps of: (a) providing alabel in the reaction chamber; where the label does not appreciably bindto any IAC or assay reagents in the assay device; (b) detecting a signalin at least one discrete zone of the diagnostic lane, where the signalis generated from the label; and (c) determining the progress and timeof completion of the assay in the assay device from at least one of: (i)a rate of change of the amount of the signal; and (ii) an absoluteamount of the signal.

The term “discrete zone” as used herein can refer to a region of anassay device. For example, if a diagnostic lane exists as a capillarytube, the capillary tube can comprise multiple discrete zones. Signalintensity can be monitored independently in each zone. An assaymeasurement can be measured in one zone on one capillary tube, and anIAC measurement can be measured in another discrete zone in anothercapillary tube, or alternatively, in the same capillary tube. Theseexamples are for illustrative purposes only and are not meant to belimiting.

The term “absolute amount of signal” as used herein can refer to ameasure of signal in an assay device. The term can refer to a signalintensity measurement at any region of an assay device relating signalintensity versus time and/or distance. A region can represent onediscrete measurement of signal intensity.

The term “rate of change of the amount of signal” as used herein canrefer to a first derivative of signal intensity. A rate of change can bedetermined at one region of a measurement relating signal intensityversus distance along a diagnostic zone. In addition, a rate of changecan be determined at one region of a measurement relating signalintensity versus time. These examples are not meant to be limiting andare for illustrative purposes only.

In a preferred embodiment, the derivative of the rate of change of theamount of the signal is determined. In another preferred embodiment, theabsolute amount of the signal is averaged. In another preferredembodiments, the rate of change is a negative rate of change of theamount of the signal.

The term “negative rate of change” as used herein can refer to adecreasing rate of change of a signal.

In a preferred embodiment, the label is linked to an MBP. In anotherpreferred embodiment, the MBP and reagents are selected from the groupconsisting of binding protein, antibody, antibody fragment, protein,peptide, and organic molecule. In other preferred embodiments, the labelis selected from the group of molecules consisting of dye, fluorescenceemitting dye, chemiluminescence emitting dye, infrared emitting dye,colloidal sol, molecule that generates an electrical and/or magneticsignal, and enzyme.

In another aspect, the invention features an apparatus for measuringprogress and time of completion for an assay in an assay device,comprising: (a) the assay device, comprising a reaction chamber and atleast one diagnostic lane; (b) an optical component; (c) a signalprocessor. A label can be provided in the reaction chamber and the labelpreferably does not appreciably bind to any assay reagents in the assaydevice. The optical component may detect a signal in at least onediscrete zone of the diagnostic lane, where the signal is generated fromthe label. The signal processor can determine the progress and time ofcompletion of the assay in the assay device from at least one of: (i) arate of change of the amount of the signal; and (ii) an absolute amountof the signal.

In yet another aspect, the invention features a kit for measuringprogress and time of completion for an assay in an assay device. The kitmay comprise: (a) at least one of a Food and Drug Administration Labeland a set of instructions; and (b) an apparatus, comprising: (i) theassay device, comprising a reaction chamber and at least one diagnosticlane; (ii) an optical component; (iii) a signal processor. A label maybe provided in the reaction chamber and the label preferably does notappreciably bind to any assay reagents in the assay device. The opticalcomponent may detect a signal in at least one discrete zone of thediagnostic lane, where the signal is generated from the label. Thesignal processor can determine the progress and time of completion ofthe assay in the assay device from at least one of: (i) a rate of changeof the amount of the signal; and (ii) an absolute amount of the signal.

IAC for Identifying Deviant Assay Results Based on Signal Intensityand/or Signal Shape

The term “deviant assay results” as used herein can refer to deviancefrom an average shape of a signal for an IAC or an assay. A signal canbe expressed as intensity versus distance in a diagnostic lane, forexample. If a shape of such a signal substantially deviates from anaverage shape of such a signal, then the assay result may be deviant. Anaverage shape of a signal can be determined from multiple measurementsof signals in different assay devices. In addition, if a shape of an IACsignal differs from a measured signal, then the measured signal may beaberrant. Like all of the IACs of the invention, this IAC can bemonitored in conjunction with any other IAC or assays of the invention.

Therefore, in one aspect, the invention features a method fordetermining deviant assay results in an assay device. The assay devicecomprises a reaction chamber and one or more diagnostic lanes. Themethod comprises the steps of: (a) providing a label in the reactionchamber; where the label does not appreciably bind to any assay reagentsin the assay device; (b) detecting an assay signal (AS) and anindependent assay control signal (IACs) in at least two discrete zonesin one or more diagnostic lanes, where the signal is generated by thelabel; and (c) determining the deviant assay result in the assay deviceby comparing an intensity and/or a shape of the AS with an intensityand/or a shape of the IACs.

The assay signal and independent assay control signal can be measured inseparate discrete zones. The discrete zones may exist in one capillarytube of an assay device, or may exist in different capillary tubes of anassay device.

In a preferred embodiment the label is linked to a MBP. In anotherpreferred embodiment, the MBP and other reagents are selected from thegroup consisting of binding protein, antibody, antibody fragment,protein, peptide, and organic molecule. In other preferred embodiments,the label is selected from the group of molecules consisting of dye,fluorescence emitting dye, chemiluminescence emitting dye, infraredemitting dye, colloidal sol, molecule that generates an electricaland/or magnetic signal, and enzyme.

In another aspect, the invention features an apparatus for determiningdeviant assay results in an assay device. The apparatus can comprise:(a) the assay device, comprising a reaction chamber and at least one ormore diagnostic lanes; (b) an optical component; and (c) a signalprocessor. A label may be provided in the reaction chamber. The labelpreferably does not appreciably bind to any assay reagents in the assaydevice. The optical component may detect an assay signal (AS) and anindependent assay control signal (IACS) in at least two discrete zonesin one or more diagnostic lanes, where the signal is generated by thelabel. The signal processor may determine the deviant assay result inthe assay device by comparing a shape of the AS with a shape of theIACS.

In yet another aspect, the invention features a kit for determiningdeviant assay results in an assay device. The kit may comprise: (a) atleast one of a Food and Drug Administration Label and a set ofinstructions; and (b) an apparatus, comprising: (i) the assay device,comprising a reaction chamber and at last one or more diagnostic lanes;(ii) an optical component; and (iii) a signal processor. A label may beprovided in the reaction chamber. The label preferably does notappreciably bind to any assay reagents in the assay device. The opticalcomponent may detect an assay signal (AS) and an independent assaycontrol signal (IACS) in at least two discrete zones in one or morediagnostic lanes, where the signal is generated by the label. The signalprocessor may determine the deviant assay result in the assay device bycomparing a shape of the AS with a shape of the IACS.

Smoothing Signals

The term “smoothing” as used herein can refer to decreasing thevariability in an assay signal. Smoothing is particularly useful forquantifying assay signals measured under assay conditions where thesignal to noise ratio is low. A particular problem associated with sucha condition is that signal processors that detect and/or quantifyincreases and decreases in signal intensity can be erroneouslytriggered. Methods of this invention that smooth signals measured insuch conditions can enhance the reliability of signal processing.Smoothing can be utilized in conjunction with any other IACs of theinvention. Methods for smoothing signals are well known in the art andare described hereafter.

The invention relates in part to any apparatus and computer programmablemedium wholly capable or partially capable of performing a smoothingmethod of the invention.

Hence, in another aspect, the invention features a method for smoothingbackground, IAC, and assay signal determinations in an assay device,where the assay device comprises a reaction chamber and at least onediagnostic lane. The method comprises the steps of: (a) providing alabel in the reaction chamber; where the label does not appreciably bindto any IAC and assay reagents in the assay device; (b) detecting thesignal in the diagnostic lane, where the signal is generated from thelabel; and (c) smoothing the signal.

In a preferred embodiment, the label is linked to a first MBP. Inanother preferred embodiment, the first MBP and other reagents can beselected from the group consisting of binding protein, antibody,antibody fragment, protein, peptide, and organic molecule. In yetanother preferred embodiment, the label is selected from the group ofmolecules consisting of dye, fluorescence emitting dye,chemiluminescence emitting dye, infrared emitting dye, colloidal sol,molecule that generates an electrical and/or magnetic signal, andenzyme.

In other preferred embodiments, the invention relates to a smoothingmethod comprising the step of providing a second MBP. The second MBP islocated in the diagnostic lane and the first MBP and the second MBP havespecific binding affinity for one another. The second MBP does notappreciably bind to any IAC and assay reagents in the assay device,except the first MBP.

In another preferred embodiment, the invention relates to a smoothingmethod comprising the step of providing a second MBP, where the secondMBP is introduced to the reaction chamber. The first MBP and the secondMBP have specific binding affinity for one another and the second MBPdoes not appreciably bind to any IAC and assay reagents in the assaydevice. The second MBP comprises an affinity tag, the diagnostic lanecomprises an affinity tag partner (ATP), and the ATP has specificbinding affinity to the affinity tag. The second MBP, the ATP, and theaffinity tag do not appreciably bind to any IAC and assay reagents inthe assay device, and the first MBP and the second MBP have specificbinding affinity for one another.

In yet another preferred embodiment, the invention relates to asmoothing method comprising the step of providing a second MBP and afirst affinity tag partner (ATP) to the reaction chamber. The first MBPand the second MBP have specific binding affinity for one another, andthe second MBP does not appreciably bind to any IAC and assay reagentsin the assay device. The second MBP comprises a first affinity tag, thefirst ATP has specific binding affinity to the first affinity tag, andthe first ATP comprises a second affinity tag. The diagnostic lanecomprises a second ATP, where the second ATP has specific bindingaffinity for the second affinity tag, and the second MBP, the first ATP,the second ATP, the first affinity tag, and the second affinity tag donot appreciably bind to any assay reagents in the assay device.

In other preferred embodiments, the smoothing comprises the step ofaveraging the signal.

In another aspect, the invention features an apparatus for smoothingassay signal determinations in an assay device. The kit may comprise:(a) the assay device, which comprises a reaction chamber and at leastone diagnostic lane; (b) an optical component; and (c) a signalprocessor. A label may be provided in the reaction chamber, where thelabel does not appreciably bind to any assay reagents in the assaydevice. The optical component may detect the signal in the diagnosticlane, where the signal is generated from the label. The signal processormay smooth the signal.

In yet another aspect, the invention features a kit for smoothing assaysignal determinations in an assay device. The kit may comprise: (a) atleast one of a Food and Drug Administration Label and a set ofinstructions; and (b) an apparatus, comprising: (i) the assay device,comprising a reaction chamber and at least one diagnostic lane; (ii) anoptical component; and (iii) a signal processor. A label may be providedin the reaction chamber, where the label does not appreciably bind toany assay reagents in the assay device. The optical component may detectthe signal in the diagnostic lane, where the signal is generated fromthe label. The signal processor may smooth the signal.

IAC for Verifying the Location of a Detection Zone

The term “verifying a location of a detection zone” as used hereinrefers to an assay and/or an IAC that can indicate whether a signaldetection apparatus is detecting a signal in an appropriate area of anassay device. For example, if an assay device is inappropriatelypositioned in a signal detection apparatus, this aspect of the inventioncan alert an operator of the deviant situation. Similarly, if an assaydevice has been manufactured inappropriately such that its detectionzones are inaccurately positioned on the assay device, this aspect canalert an operator of the deviant situation. Other factors and methodsfor verifying the location of a detection zone are described hereafter.

The term “detection zone” as used herein can refer to any region of anassay device in which a signal can be detected. This signal can extendto IAC signals, signals that will be smoothed, and assay measurementsignals, background signals, and any other type of detectable signal.

Another aspect of the invention features a method for verifying alocation of a detection zone in an assay device, where the assay devicecomprises a reaction chamber and at least one diagnostic lane. Themethod comprises the steps of: (a) providing a label in the reactionchamber, where the label does not appreciably bind to any IAC and assayreagents in the assay device; (b) measuring for a signal in at least onediscrete zone of the diagnostic lane, where the signal is generated bythe label; and (c) verifying the location of the detection zone by adetection of the signal in the discrete zone of the diagnostic lane.

In a preferred embodiment, the label is linked to an MBP. In anotherpreferred embodiment, the MBP and other reagents are selected from thegroup consisting of binding protein, antibody, antibody fragment,protein, peptide, and organic molecule. In yet another preferredembodiment the label is selected from the group of molecules consistingof dye, fluorescence emitting dye, chemiluminescence emitting dye,infrared emitting dye, colloidal sol, molecule that generates anelectrical and/or magnetic signal, and enzyme.

In another aspect, the invention features an apparatus for verifying alocation of a detection zone in an assay device. The apparatus maycomprise: (a) the assay device, where the assay device comprises areaction chamber and at least one diagnostic lane; (b) an opticalcomponent; and (c) a signal processor. A label can be provided in thereaction chamber; where the label does not appreciably bind to any assayreagents in the assay device. A signal may be measured by the opticalcomponent in at least one discrete zone of the diagnostic lane, wherethe signal is generated by the label. The location of the detection zonecan be determined by the signal processor by a detection of the signalin the discrete zone of the diagnostic lane.

In yet another aspect, the invention features a kit for verifying alocation of a detection zone in an assay device. The apparatus maycomprise: (a) at least one of a Food and Drug Administration label and aset of instructions; and (b) an apparatus, comprising: (i) the assaydevice, where the assay device comprises a reaction chamber and at leastone diagnostic lane; (ii) an optical component; and (iii) a signalprocessor. A label can be provided in the reaction chamber; where thelabel does not appreciably bind to any assay reagents in the assaydevice. A signal may be measured by the optical component in at leastone discrete zone of the diagnostic lane, where the signal is generatedby the label. The location of the detection zone can be determined bythe signal processor by a detection of the signal in the discrete zoneof the diagnostic lane.

Correction of Assay Measurements by Utilizing IACs

The invention teaches methods, computer programmable media, andapparatus useful for correcting assay measurements by utilizing IACsprovided herein. As discussed previously, assay results can deviate dueto variations in parameters that an assay operator cannot easilycontrol, such as variations in assay temperature and variations insample viscosity and chemical and biochemical composition.

Measurement of an IAC of the invention can be utilized to correct ameasured assay result so that uniform assay results can be measureddespite variations in conditions that are beyond an assay operator'scontrol. Mathematical derivations for correcting measured assay resultsare described hereafter with respect to utilizing one, two, or multipleIACs in a single assay.

Different methods, computer programmable media, and apparatus can beutilized to correct assay measurements with one or more IACmeasurements. The type of method, computer programmable medium, andapparatus used to correct an assay measurement can depend upon therelation of particular parameters of an assay. For example, therelationship between (i) a constant (β) that modifies deviations in IACmeasurements and (ii) mean values for assay measurements, can determinewhich methods, computer programmable media, and apparatus are used tocorrect an assay measurement. Specifically, certain methods, computerprogrammable media, and apparatus may be utilized if these twoparameters are related in non-linear manner, while other methods,computer programmable media, and apparatus may be utilized if these twoparameters are related in a linear manner. These parameters are definedin more detail hereafter.

The term “constant” as used herein can refer to any constant that ismultiplied by a measurement or determination of the invention. Ameasurement of the invention can be an IAC deviation or a mean value ofmultiple assay measurements, for example. As mentioned previously, IACdeviations can be multiplied by a constant in methods useful forcorrecting a measured assay result. Some constants utilized herein arereferred to as β and Γ. β and Γ can be functions of test values and IACvalues. However, in the teachings that follow, β and Γ can be solved atspecific values for test assay measurements and IAC measurements andtherefore can be referred to as constants. β can be determined, forexample, (i) by guessing a value of the constant, (ii) by solving for aslope value of a linear regression relating assay measurement deviationsand control measurement deviations, (iii) by solving a matrix functionfor a matrix consisting of assay measurement deviations and controlmeasurement deviations, and (iv) by solving equations that maximize acoefficient of correlation and minimize the quotient of the standarddeviation and the mean value of corrected assay measurements. Multipleexamples of mathematical means for solving matrix functions andcoefficients of correlations are well known to a person of ordinaryskill in the art. See, e.g., Matrixes and Tensors in Physics (2^(nd)edition), A. W. Joshi, and Probability Distributions: An Introduction toProbability Theory with Applications, 1972, C. P. Tsokos. Applicationsinvolving linear regression analysis and matrix function analysis arealso described in more detail hereafter. Γ can be determined, forexample, from the slope of a linear regression analysis for a plotrelating β and mean value of assay measurements. Each β may bedetermined from a set of assay devices. Each mean value of assaymeasurements can be determined from a set of assay devices. Thesubscript “i” is sometimes used herein to denote a discrete set of assaydevices.

Utilization of IACs When a Constant That Modifies Deviations in IACMeasurements Varies Non-linearly with Mean Values of Assay Measurements

The invention relates in part to methods, computer programmable media,and apparatus that are useful for determining corrected assay resultsfrom measured assay results under assay conditions where a constant (β)that modifies deviations in IAC results varies in a non-linear fashionwith a mean value of multiple assay measurements. Mathematical equationsdepicting the relationship between corrected assay results, measuredassay results, IAC results, and constants that modify IAC results aredefined hereafter, with respect to these conditions.

When IACs are dependent upon one another, the constant can be determinedby solving a matrix function comprising assay result measurements andIAC measurements. When IACs are not dependent upon one another, theconstant can be determined by solving for the slope of a linearregression analysis of a plot relating deviations in IAC measurementsand deviations in assay measurements. Deviations in assay and IACmeasurements can be determined from measurements using multiple assaydevices. Discrete mathematical equations and corresponding experimentalmethods can be determined for assay devices that employ multiple IACs,two IACs, or one IAC for the correction of an assay result. Thesemathematical equations are defined hereafter.

The mathematical operations described herein, including determinationsof sums, differences, products, and quotients are well known to personof ordinary skill in the art. Additionally, linear regression analysis,solving matrix algebra functions, and the creation of plots, aretechniques well known to a person of ordinary skill in the art.

The term “difference” and “differences” as used herein can refer tomathematical functions in which one quantity is subtracted from another.The quantity that is subtracted from another is independent of themanner in which the difference is expressed herein. For example, thedescription “the difference between A and B” can be expressed as “Asubtracted from B” and “B subtracted from A.” The latter interpretationis preferred for the purposes of the invention.

The term “quotient” as used herein can refer to mathematical functionsin which one quantity is divided by another quantity. The quantity thatis divided by another quantity is independent of the manner in which thequotient is expressed herein. For example, the description “the quotientbetween A and B” can be expressed as “A divided by B” and “B divided byA.” The former interpretation is preferred for the purposes of theinvention.

The term “multiple measurements” as used herein can refer to a number ofassay and/or IAC measurements that may be utilized to correct an assaymeasurement. The subscript “i” can be utilized to denote each assaydevice. The number of assay devices can be variable with respect to aparticular type of assay measurement as well as a particular IAC.Although the mathematical relations provided by the invention relate toan infinite number of assay devices, the number of assay devicespreferably span from two to one million. For example, if one type ofassay measurement is determined in ten assay devices, the measurement inthe fifth device can be referred to as T_(i) where i is equal to five.As another example, and as described hereafter, assay measurements canbe determined in multiple assay devices, a mean value of assaymeasurements can be determined, a range of assay deviations can becalculated by subtracting the calculated mean value from each assaymeasurement, and constants can be determined from plots of these assaydeviations. The subscript “i” can reflect each assay device as well aseach IAC utilized to generate multiple assay measurements as well as IACmeasurements.

A discrete IAC can be defined by the subscript “j.” The mathematicaloperations provided herein allow for an infinite number of discrete IACsto be utilized for the correction of an assay signal. However, less thanten IACs are preferably measured in an assay device for the correctionof an assay result. In an example, if ten IACs are measured in one assaydevice, the fifth IAC measured in each assay device can be referred toas IAC_(j), where j is equal to 5. Each IAC deviation for each discreteIAC can be modified by a constant to correct a measured assay result.

The terms “IAC deviation” and “δIAC” as used herein can refer to amathematical difference between a mean value of multiple IACmeasurements for each discrete IAC in an assay device and the multipleIAC measurements themselves. Multiple IAC measurements can be determinedin multiple assay devices. The multiple IAC measurements can beperformed previous to the assay measurement that may be corrected.

The terms “assay measurement deviations” and “δT” as used herein canrefer to a mathematical difference between a mean value of multipleassay measurements and the multiple assay measurements themselves. Themultiple assay measurements can be determined in multiple assay devices.The multiple assay measurements can be performed previous to the assaymeasurement that may be corrected.

The term “matrix function” as used herein can refer to a mathematicalmatrix function or an inverse matrix function. Matrix functions andinverse matrix functions are described in more detail hereafter.

Thus in one aspect, the invention features a method for determining acorrected assay result (T_(c)) from a measured assay result (T_(m)) andmultiple (j) measured control assay results (IAC_(j)). The methodcomprises the steps of: (a) measuring the T_(m) and each IAC_(j) in anassay device; (b) determining a difference between each IAC_(j) and amean value (IAC_(jave)) of multiple IAC_(j) measurements (IAC_(ji)); (c)determining a product by multiplying each difference of step (b) by aconstant (β_(j)); (d) determining a sum of every j^(th) product; and (e)subtracting the sum from T_(m).

In a preferred embodiment, each β_(j) is determined by a matrix functioncomprising each δT and each δIAC_(j). In another preferred embodiment,each β_(j) is determined from a slope of a linear regression of a plot,where the plot is differences (δT_(i)) between multiple assay resultmeasurements (T_(i)) and a mean value of T_(i) (T_(ave)) versusdifferences between IAC_(ji) and IAC_(jave).

In yet another preferred embodiment, the method is performed when oneIAC is utilized to correct an assay result. In other preferredembodiments, two IACs are utilized to correct an assay result. A personof ordinary skill in the art can readily adapt the methods, computerprogrammable media, apparatus, and mathematical relations taught hereinto effect these preferred embodiments.

In another aspect, the invention features a computer programmable mediumembodying a program of instructions for causing a processor to perform amethod for determining a corrected assay result (T_(c)) from a measuredassay result (T_(m)) and multiple (j) measured control assay results(IAC_(j)). The method comprises the steps of: (a) measuring said T_(m)and each IAC_(j) in an assay device; (b) determining a differencebetween each IAC_(j) and a mean value (IAC_(jave)) of multiple IAC_(j)measurements (IAC_(ji)); (c) determining a product by multiplying eachdifference of step (b) by a constant (β_(j)); (d) determining a sum ofevery j^(th) product; and (e) subtracting the sum from T_(m).

In yet another aspect, the invention features an apparatus useful forperforming the method defined in the previous paragraph.

Preferred embodiments of the invention relate to utilizing any signalmeasurements and IAC measurements described herein for use in methods,computer programmable media, and apparatus useful for correcting ameasured assay result.

Utilization of IACs When a Constant That Modifies Deviations in IACMeasurements Varies Linearly with Mean Values of Assay Measurements

The invention relates in part to methods, computer programmable media,and apparatus that are useful for determining corrected assay resultsfrom measured assay results under assay conditions where a constant (β)that modifies deviations in IAC results varies in a linear fashion witha mean value of multiple assay measurements. Mathematical equationsdepicting the relationship between corrected assay results, measuredassay results, IAC results, and constants that modify IAC results aredefined hereafter, with respect to these conditions.

As described previously, when IACs are dependent upon one another, theconstant can be determined by solving a matrix function comprising assayresult measurements and IAC measurements. When IACs are not dependentupon one another, the constant can be determined by solving for theslope of a linear regression analysis of a plot relating deviations inIAC measurements and deviations in assay measurements. Deviations inassay and IAC measurements can be determined from measurements usingmultiple assay devices. Discrete mathematical equations andcorresponding experimental methods can be determined for assay devicesthat employ multiple IACs, two IACs, or one IAC for the correction of anassay result. These mathematical equations are defined hereafter.

Hence, in one aspect, the invention features a method for determining acorrected assay result (T_(c)) from a measured assay result (T_(m)) andmultiple (j) measured control assay results (IAC_(j)). The methodcomprises the steps of: (a) measuring the T_(m) and each IAC_(j) in anassay device; (b) determining a difference (δIAC_(j)) between eachIAC_(j) and a mean value (IAC_(jave)) of multiple IAC_(j) measurements(IAC_(ji)); (c) determining a product by multiplying each difference ofstep (b) by a constant (T_(j)); (d) determining a sum of the integer 1and every j^(th) product; (e) determining a quotient between the sum andthe T_(m).

In a preferred embodiment each Γ_(j) is a slope of a linear regressionof a plot, where the plot is multiple β_(j) determinations versus meanvalues (T_(ave)) of multiple assay result measurements (T_(i)). Inanother preferred embodiment, each β_(j) is determined by a matrixfunction comprising each δT and each δIAC_(j). In other preferredembodiments, each β_(j) is determined from a slope of a linearregression of a plot, where the plot is differences (δT) betweenmultiple assay result measurements (T_(i)) and a mean value of T_(i)(T_(ave)) versus differences between IAC_(ji) and IAC_(jave).

In a preferred embodiment, the method is performed when one IAC isutilized to correct an assay result. In another preferred embodiment,two IACs are utilized to correct an assay result. A person of ordinaryskill in the art can readily adapt the methods and mathematicalrelations taught herein to effect these preferred embodiments.

In another aspect, the invention features a computer programmable mediumembodying a program of instructions for causing a processor to perform amethod for determining a corrected assay result (T_(c)) from a measuredassay result (T_(m)) and multiple (j) measured control assay results(IAC_(j)). The method comprises the steps of: (a) measuring the T_(m)and each IAC_(j) in an assay device; (b) determining a difference(δIAC_(j)) between each IAC_(j) and a mean value (IAC_(jave)) ofmultiple IAC_(j) measurements (IAC_(ji)); (c) determining a product bymultiplying each difference of step (b) by a constant (Γ_(j)); (d)determining a sum of the integer 1 and every j^(th) product; (e)determining a quotient between the sum of multiple entities and theT_(m).

In yet another aspect, the invention features an apparatus useful forperforming the method described in the previous paragraph.

Other methods exist for correcting an assay result when a constant thatmodifies deviations in IAC results varies in a linear fashion with amean value of multiple assay measurements. Hence, in another aspect, theinvention features a method for determining a corrected assay result(T_(c)) from a measured assay result (T_(m)) and multiple (j) measuredcontrol assay results (IAC_(j)). The method comprises the steps of: (a)measuring the T_(m) and each IAC_(j) in an assay device; (b) determininga function of each IAC_(j); (c) determining a sum of the integer 1 andeach function; and (d) determining a quotient between T_(m) and the sum.

The term “function” as used herein can refer to a mathematical operationthat can be expressed in terms of a power series expansion ofdifferences (δIAC_(j)) between each IAC_(j) and a mean value(IAC_(jave)) of multiple IAC_(j) measurements (IAC_(ji)). A power seriesexpansion is well known to a person of ordinary skill in the art.

In a preferred embodiment, the method is performed when one IAC isutilized to correct an assay result. In another preferred embodiment,two IACs are utilized to correct an assay result. A person of ordinaryskill in the art can readily adapt the methods and mathematicalrelations taught herein to effect these preferred embodiments.

In another aspect, the invention features a computer programmable mediumembodying a program of instructions for causing a processor to perform amethod for determining a corrected assay result (T_(c)) from a measuredassay result (T_(m)) and multiple (j) measured control assay results(IAC_(j)). The method comprises the steps of: (a) measuring the T_(m)and each IAC_(j) in an assay device; (b) determining a function of eachIAC_(j); (c) determining a sum of the integer 1 and each function; and(d) determining a quotient between T_(m) and the sum.

In yet another aspect, the invention features an apparatus that isuseful for performing the method defined in the previous paragraph.

When one IAC is measured, another method can be utilized to correct anassay measurement when IAC measurements vary proportionately with assaymeasurements.

Hence, in another aspect, the invention features a method fordetermining a corrected assay result (T_(c)) from a measured assayresult (T_(m)) and a measured independent assay control result (IAC).The method comprises the steps of: (a) measuring the T_(m) and the IACin an assay device; (b) determining a quotient between a mean value(IAC_(ave)) of multiple IAC measurements (IAC_(i)) and the IAC; and (c)determining a product by multiplying the quotient of step (b) by saidT_(m).

In another aspect, the invention features a computer programmable mediumembodying a program of instructions for causing a processor to perform amethod for determining a corrected assay result (T_(c)) from a measuredassay result (T_(m)) and a measured independent assay control result(IAC). The method is identical to that defined in the previousparagraph.

In yet another aspect, the invention features an apparatus useful fordetermining a corrected assay result (T_(c)) from a measured assayresult (T_(m)) and a measured independent assay control result (IAC).The apparatus is useful for performing the method defined in theprevious paragraph.

As determined previously, preferred embodiments of the invention canrelate to utilizing any signal measurements and IAC measurementsdescribed herein for use in methods, computer programmable media, andapparatus useful for correcting a measured assay result.

Correcting Assay Measurements by Utilizing Apparatus

The invention relates in part to methods for correcting assaymeasurements by measuring IACs in apparatus. Any of the IAC andsmoothing methods, apparatus, and kits defined herein can be utilizedfor the correction of assay measurements. Any combination of IACs andsmoothing methods, apparatus, and kits can be combined or utilizedindividually. For example, an apparatus useful for smoothing signals andan apparatus useful for correcting an assay measurement by monitoringthe rate of flow can exist as one apparatus or can be combined into oneapparatus useful for correcting assay measurements.

Embodiments of the invention relate to apparatus that can measure assaysignals and/or IAC signals by an optical device within the apparatus.Optical apparatus are defined in U.S. patent application Ser. No.09/003,090, entitled “Immunoassay Fluorometer,” Buechler et al., filedJan. 5, 1998, now U.S. Pat. No. 6,830,731.

In a preferred embodiment, an apparatus of the invention comprises amedia carrier. The term “media carrier” is fully defined in U.S. patentapplication Ser. No. 09/003,066, entitled “Media Carrier for an AssayDevice,” Buechler et al., filed Jan. 5, 1998, now U.S. Pat. No.6,074,616. A media carrier may comprise computer programmable mediadefined herein.

Hence, the invention relates in part to apparatus that are capable of:(i) measuring an assay signal in an assay device, and (ii) measuring oneor more IAC signals defined herein, (iii) smoothing the signals, and(iv) correcting the assay result by utilizing the IAC signal or signals.The assay signal and IAC signal or signals can be measured using anoptical component of the apparatus and the signals can be smoothed by aprocessor or co-processor component of the apparatus. The processor orany co-processor can correct the assay measurement according to the IACsignal or signals. The processors or co-processors of the apparatus canoperate in conjunction with one or more media carriers.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, as well asfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

Certain figures illustrate signal measurement for particular embodimentsof the invention.

FIG. 1 is a time dependent profile of an integrated CKMB assay signal.

FIG. 2 is a time dependent profile of an integrated troponin I assaysignal.

FIG. 3 is a time dependent profile of an integrated myoglobin assaysignal.

Other figures illustrate particular embodiments of IACs of theinvention.

FIG. 4 is a time dependent profile of the integrated timegate IACsignal.

FIG. 5 is a time dependent profile of an integrated flow IAC signal.

FIG. 6 is a time dependent profile of an integrated non-specific bindingIAC signal.

FIG. 7 is a time dependent profile of a timing IAC signal.

FIG. 8 is a time dependent profile of an IAC concerning human chorionicgonadotrophin (hCG) signals.

FIG. 9 is a time-dependent profile of normalized hCG assay signals.

Some figures illustrate embodiments of signal processing IACs of theinvention.

FIG. 10 depicts specific time zones in an assay device diagnostic lane.

FIG. 11 illustrates zone determination and identification in adiagnostic lane of an assay device.

FIG. 12 depicts the effects of bidirectional filtering functions.

FIG. 13 illustrates filtering effects as a function of time constants.

FIG. 14 depicts filtering effects as a function of parabolic fitfunctions.

FIG. 15 illustrates filtering effects with respect to maximum allowedrate of change functions.

FIG. 16 depicts filtering effects with respect to rejection zones.

FIG. 17 illustrates filtering effects with respect to second derivativeanalysis.

All of these embodiments are discussed in detail hereafter.

Various embodiments of the invention may be implemented using computerhardware, software or a combination thereof and may be implemented in acomputer system or other processing system. An example computer systemis illustrated in FIG. 18.

DETAILED DESCRIPTION OF THE INVENTION

This invention is related to novel methods that utilize independentassay controls (IAC). The IAC communicate optically with an instrumentfor the definition of the status of an immunoassay in an assay device.The IAC are related to the visualization and interpretation ofimmunoassays in an assay device that are specific, independent bindingevents and they can be related to the visualization and interpretationof total assay device fluorescence which can be related to non-specificbinding events. In one embodiment, the IAC are used for defining thestatus and the time of completion of an immunoassay in an assay device.In another embodiment, the IAC define zones and profiles of assaysignals. In yet another embodiment, the IAC redefine assay signals forpurposes of correction of the assay signals as a function of samplematrix differences and sample flow rates in assay devices.

The IAC are immunoassays that are performed by the assay device. Themagnitudes of the IAC signals are predictable within a range, set by themanufacturer, unlike the magnitudes of the analyte signals which areunpredictable. The novel features of the IAC are that they areindependent of the presence or amount of analyte but dependent on thematrix of the sample and the progress of the immunoassay process in theassay device. The independent nature of the IAC from the analyte assaysallows the design of their use to calibrate analyte assay signals thatcan be dependent on sample matrix and to define the shape and magnitudeof the fluorescent signal to verify that the immunoassay process hastaken place without error.

This invention relates to the use of IAC for an accurate and reliablemeasurement of analyte concentrations. In general, analytes are measuredusing methods related to immunoassays. One skilled in the art willrecognize that immunoassays can be performed in non-competitive andcompetitive formats and the inventive teachings described herein areapplicable to these formats but are not limited solely to these formats.For example, U.S. Pat. Nos. 5,028,535 and 5,089,391 teach a form ofimmunoassay that incorporates a threshold response with respect to thegeneration of signal as a function of analyte concentration and thismethodology can be utilized with the IAC methodology. The methodsutilize, for example, assay devices incorporating lateral flow ofreagents and sequential binding of reagents to a solid surface, as wellas instruments, which are described in U.S. Pat. No. 5,458,852 andco-pending U.S. patent application Ser. No. 08/458,276, Nowakowski etal., “Devices for Ligand Receptors Methods,” filed Jan. 27, 1995; U.S.patent application Ser. No. 08/065,528, Buechler, “Diagnostic Devicesand Apparatus for the Controlled Movement of Reagents WithoutMembranes,” filed May 21, 1992; U.S. patent application Ser. No.08/447,895, Buechler, “Diagnostic Devices and Apparatus for theControlled Movement of Reagents Without Membranes,” filed May 23, 1995;U.S. patent application Ser. No. 08/447,981, Buechler, “DiagnosticDevices and Apparatus for the Controlled Movement of Reagents WithoutMembranes,” filed May 23, 1995; U.S. patent application Ser. No.08/810,569, Buechler, “Diagnostic Devices and Apparatus for theControlled Movement of Reagents Without Membranes,” filed Feb. 28, 1997;U.S. patent application Ser. No. 08/828,041, Buechler, “DiagnosticDevices and Apparatus for the Controlled Movement of Reagents WithoutMembranes,” filed Mar. 27, 1997; all of which are incorporated byreference in their entirety including all figures, tables, and drawings.One skilled in the art will recognize that with the inventive teachingsdescribed herein, other assay devices and instruments can also utilizethese novel methods.

The reagents comprising the IAC are designed to provide informationregarding the status of an immunoassay in an immunoassay device. Thereagents are paired to result in a binding reaction as a result of theimmunoassay, as one skilled in the art will recognize as anantibody/antigen binding reaction. The reagents comprising the IAC canbe binding proteins, for example, antibodies and binding fragments thatbind specifically to proteins, peptides, ligands and ligand analogues.The reagents can be in solution or attached to a solid phase. Thereagents can be modified so that an antibody can bind to a ligandattached to another antibody or protein. As a result of the assayprocess, at least two reagents bind, either in solution or on a solidphase or both. At least one of the reagents of the IAC is attached to alabel, directly or indirectly as a result of the assay process.Attachment can be made through the use of covalent bonds, electrostaticinteractions, hydrophobic interactions and one skilled in the art isfamiliar with these techniques. A label can be dyes, fluorescent dyes,colloidal sols, molecule that generates an electrical and/or magneticsignal(s), and enzymes that convert molecules into dyes or reagents thatbecome charged as a result of the assay process. In a preferredembodiment, the label is comprised of fluorescent dyes. In aparticularly preferred embodiment, the fluorescent dyes are imbibed intoor attached to particles, and they excite at about 670 nm and emit atabout 760 nm. See, e.g., European Patent Application 94931287.0,Buechler et al., “Fluorescence Energy Transfer and Intramolecular energytransfer in particles using novel compounds,” filed Sep. 23, 1994; U.S.patent application Ser. No. 08/601,492, Buechler, “Fluorescence EnergyTransfer in Particles,” filed Feb. 14, 1996; U.S. patent applicationSer. No. 08/274,534, Buechler et al., “Fluorescence Energy Transfer inParticles,” filed Jul. 12, 1994; and U.S. patent application Ser. No.08/311,098, Buechler et al., “Fluorescence Energy Transfer andIntramolecular Energy Transfer in Particles Using Novel Compounds,”filed Sep. 23, 1994; all of which are incorporated herein by referencein their entirety including all figures, tables, and drawings. The sizesof particles useful for this invention vary between about 2 nm to 4000nm, and preferably between about 50 nm and 300 nm. In the case ofutilizing enzymes to convert molecules into a label, one skilled in theart recognizes that conjugates of enzymes and, for example, antibodies,can be made using heterophilic cross linking reagents.

An important aspect of the invention is that the IAC reagents reside onseparate or independent conjugates from each other and from the reagentsused in performing the analyte assays. This critical function allows theIAC reagents to be independent of the analyte assay reagents so that theconcentration of the analyte in the assay does not affect the perceivedoutcome of the assay as defined by the IAC. In addition, separating theIAC conjugates allows a novel approach to developing controls that havea variety of functions, each independent of each other, for defining thestatus of an immunoassay in a device, particularly in a device where thereagents move in a lateral fashion and the binding of the labeledconjugates to the solid phase occurs in a sequential manner. Forexample, the IAC conjugates and the assay conjugates are uncoupledbecause one may need different dynamic ranges for the analyte assays.For example, assay dynamic ranges in an assay device for myoglobin wouldbe 0 to 1000 ng/ml, creatine kinase would be 0 to 150 ng/ml andtroponin, which requires high sensitivity, would be 0 to 10 ng/ml. Thesubstantially different dynamic ranges of each of these assays wouldrequire that, for example, different particle sizes of the label beused. Large dynamic range assays, such as myoglobin, would use small butmany particles, whereas shorter dynamic range assays that require highsensitivity, such as troponin, would use larger and fewer particles. Oneskilled in the art recognizes this approach to design of immunoassaysand furthermore, the use of enzyme conjugates with varying specificactivities also creates assays with different dynamic ranges. Thus, dueto the constraints of immunoassay design for the analytes, the IAC andassay conjugates must be uncoupled to prevent interferences. In anotherexample of the versatility of the IAC and the necessity for uncouplingthe binding events of the IAC and the assay reagents, analyteimmunoassays and IAC that function in devices by the lateral movement ofthe assay reagents can potentially interfere with each other. Forexample, the binding of a label to a solid phase zone up stream from thebinding of the same label to a down stream solid phase in a diagnosticlane can affect the signal of the down stream binding because theupstream binding event will remove all or a fraction of the reagent.Thus, the upstream binding event will affect the down stream bindingevent resulting in analyte assays or IAC results that cannot bepredictable. The novel methods described herein provide that the IAC andanalyte assay reagents be both separate and independent of the bindingof each other and the assay reagents to solid phases, which in turn,allow reproducible and predictable signals to be generated for both theanalyte assays and the IAC in lateral flow devices.

The IAC function in an assay device and communicate with an instrument.In a preferred embodiment, the instrument is a portable, battery poweredfluorometer that incorporates a means for measuring an optical signalfrom an assay device and transforms the optical signal into anelectrical signal. In a preferred embodiment, the instrument utilizes alaser diode emitting light at about 670 nm and a silicon photodiode asthe excitation source and detector, respectively. The instrument alsoincorporates a means for storage of a variety of routines that allow itto measure and calculate the presence or concentration of one or moreanalytes, either utilizing information from the IAC or not, and tointerface with outside data systems. A preferred instrument is describedin U.S. patent application Ser. No. 09/003,090 entitled “ImmunoassayFluorometer,” Buechler et al., filed Jan. 5, 1998, now U.S. Pat. No.6,830,731, and U.S. patent application Ser. No. 09/003,066 entitled“Media Carrier for an Assay Device,” Buechler et al., filed Jan. 5,1998, now U.S. Pat. No. 6,074,616, both of which are incorporated byreference herein. One skilled in the art will recognize that instrumentsincorporating means for measuring the reflectance or absorbance of alight absorbing compound, that is, a label, can also be used in thepractice of the inventive teachings described herein.

In a preferred embodiment, the assay device performs binding events,specific and non-specific for the IAC and one or more immunoassays forthe measurement of the presence or concentration of one or moreanalytes. The immunoassay device comprises reagents for the IAC and forthe measurement of analytes. The immunoassay device is generally aone-step device where a user applies one or more samples to one or aplurality of devices and the immunoassay process is performed within thedevice. The sample flows through the device in a lateral fashion withoutuser interface utilizing capillarity as the driving force for fluidmovement. In this preferred embodiment, the assay device generally bindsa fluorescent label to a non-bibulous surface of the device as a resultof the assay process. One skilled in the art will recognize that thebinding of the variety of labels as a result of the assay process canalso take place on bibulous supports, for example, in membranes and inpaper. In a preferred embodiment, the binding of the label to a surfaceas a result of the immunoassay process can take place in one or aplurality of zones on the diagnostic lane of the assay device.

The immunoassay device generally has a compartment or chamber and adiagnostic lane in which and onto which reagents of the assay processare placed. In a preferred embodiment, a reaction chamber, which definesthe volume of the reaction mixture, contains the reagents of the IAC andof the analyte specific assay, for example, fluorescent antibodyconjugates. In assay devices that do not comprise bibulous supports, thecapillary driving force is defined by the texture of the surfaces andthe proximity of two surfaces, a base and a lid, that create a capillaryspace. A reaction chamber therefore has two surfaces onto which reagentsof the IAC and analyte assays can be placed. In assay devices thatcomprise bibulous supports, such as membranes, the capillary drivingforce for fluid flow is generally defined by the pore size of themembrane. The diagnostic lane is the means in the assay device thatbinds the label in one or more discrete zones as a result of the assayprocess.

In a preferred embodiment the immunoassay process comprises adding asample to an assay device, where the sample flows through a filter or amesh for purposes of filtering cells from plasma or filtering outdebris, or for lysing cells, respectively. A portion of the sample flowsinto a reaction chamber and combines with the assay reagents to form areaction mixture. The reaction mixture is usually allowed to incubatefor a period of time, generally about 30 seconds to about 5 min, toallow the reactions to reach substantial equilibrium binding. Thereaction mixture then continues to flow in a lateral fashion, past oneor more discrete zones on a solid phase of the diagnostic lane, wherebythe label in the reaction mixture binds to one or more of the zones as aresult of the assay process. Excess sample flowing behind the reactionmixture acts as a wash to remove label that has not bound to the solidphase. The extent and degree of label from the analyte conjugates andfrom the conjugates of the IAC binding to the solid phase in the zone orzones can be indicative of the concentration of analyte in the sampleand the flow mechanics, incubation time, reaction mixture homogeneityand sample matrix, respectively. The concentrations of the analytes andthe intensities of the IAC are quantified by the instrument by placingthe assay device into the instrument and measuring the light energyresulting from the binding of the labels to the solid phase.

The proximity of the IAC reagents in the reaction chamber can define andmonitor functions related to the non-specific binding of label, flowmechanics, incubation time, homogeneity of the reaction mixture andsample matrix of the assay.

In a preferred embodiment, a non-specific binding control IAC isincorporated in the device. This function is termed the non-specificbinding control. The non-specific binding control is comprised of theentire repertoire of label in the device and a zone or zones on thediagnostic lane. The zone or zones of the diagnostic lane can comprisean antibody or the surface of the device or both that does notspecifically bind the label but binds the label through non-specificbinding events. One skilled in the art is familiar with non-specificbinding events. Thus, the non-specific binding control IAC monitors thedegree of non-specific binding of the label to the solid phase andidentifies problems with sample matrices. For example, the presence ofheterophilic antibodies in samples can crosslink label antibodyconjugates to other antibodies and this non-specific binding event canresult in an increase in label binding to the solid phase zones of theassay device.

In a preferred embodiment, a flow control IAC is incorporated in thedevice and this function is termed a flow control. The flow control IACis comprised of a label onto which is attached proteins, antibodies,ligands, peptides, or ligand analogues and can be placed on the base orlid of the reaction chamber with the assay reagents. The flow controlIAC reagents are reconstituted in the reaction chamber by the sample andultimately bind to the solid phase of the diagnostic lane. The solidphase reagent is complementary with the reagent on the flow controllabel such that the binding can take place. For example, a ligandanalogue is attached to the label and an antibody to the ligand analogueis attached to the solid phase. The flow control IAC ultimatelydocuments the flow rate of the reaction mixture in the capillary of thediagnostic lane. Preferred reagents for the flow control are fluorescentparticles about 50 nm to 300 nm in diameter and have attached anantibody, a ligand or a ligand analogue. The solid phase reagent ispreferably a ligand, ligand analogue or an antibody, respectively. Oneskilled in the art will recognize that various combinations of bindingevents with reagents can result to define the flow control.

In another preferred embodiment, the incubation time of the reactionmixture in the reaction chamber is monitored by the IAC and is termed atime gate control. For example, in this embodiment, a label with aligand or a ligand analogue covalently attached, is placed on the baseof the reaction chamber and an antibody with a tag covalently attachedthat binds the ligand or ligand analogue of the label is placed on thelid of the reaction chamber. The labeled ligand conjugate is termed atime gate control label and the antibody tag conjugate is termed a timegate control antibody. When sample flows into the reaction chamber as aresult of the assay, the lid and base reagents are reconstituted by thesample and the lid antibody and the label begin to bind to each other.The binding of these two reagents of the IAC is a function of time,their relative dissolution rates and their relative diffusioncoefficients. The time element of the binding can therefore monitor theincubation time of the reaction mixture. Thus, what is formed in thisreaction mixture is a label bound with an antibody tag. The tag of theantibody ultimately binds to an antibody on the solid phase of thediagnostic lane and the intensity of the signal from the label isrelated to the incubation conditions of the reagents in the reactionchamber. Preferred labels for the time gate control are fluorescentparticles about 50 nm to 300 nm in diameter and have attached anantibody or a ligand or a ligand analogue. Preferred complementaryreagents that bind to the label during the incubation are proteins, suchas bovine serum albumin that have a ligand complementary to the antibodyor an antibody complementary to the ligand or the ligand analogue,respectively. Preferred tags are ligands, ligand analogues, peptides andthe like. One skilled in the art will recognize that variouscombinations of ligands, ligand analogues, antibodies and peptides, bothin solution and on the solid phase, can be configured to provide a timegate control. One skilled in the art will also recognize that the timegate control label and time gate control antibody can be placed in anumber of different locations in the device. For example, the time gatecontrol antibody can be added to the sample or placed in the deviceprior to entering the reaction chamber, such as in the sample or in afilter, respectively, and the label can be placed on the base and/or thelid of the reaction chamber.

In another preferred embodiment, a label with a ligand analogue and anantibody1 with a tag1 are placed on the base of the reaction chamber andan antibody2 with a tag2 is placed on the lid. In this preferredembodiment, the congruency of the reaction mixture is defined withrespect to the homogeneity of the reaction mixture as it relates to therelative dissolution of the reagents on the base and the lid. Thiscontrol is termed the reaction chamber control. The reagents arereconstituted in the reaction chamber with sample, and the rate at whichsample flows into the reaction chamber can affect the relativedissolution of the regents. For example, if the sample flows slowly intothe reaction chamber, more of the reagents are dissolved by theadvancing sample front than if flow into the chamber is rapid. Therelative dissolution of the reagents on both lid and base should thus beoptimized so that dissolution is independent of flow rate into thechamber. However, this ideal situation is not always possible to achievein practice. Thus, the lid antibody2 tag2 binds to the ligand analogueof the label on the base and the base antibody1 tag1 binds to the tag2of the lid antibody2 tag2. This binding sequence comes to equilibrium inthe reaction mixture that is congruent with the dissolution of the lidand the base reagents. The tag1 of the base antibody1 ultimately bindsto an antibody on the solid phase of the diagnostic lane resulting inthe binding of the label to the solid phase. The relative signal of thisIAC is thus dependent on the homogeneity of the reaction mixture in thereaction chamber. Preferred reagents for the reaction chamber controlare fluorescent particles about 50 nm to 300 nm in diameter and haveattached an antibody, a ligand or a ligand analogue. The complementarycontrol reagent in solution with the label and on the lid are preferablya protein with a ligand or ligand analogue attached, or antibodiescomplementary to the ligand or ligand analogue. The solid phase reagentis preferably a ligand, ligand analogue or an antibody.

A preferred embodiment of the invention utilizes the IAC for definingthe progress and the time of completion of the immunoassay in an assaydevice. The timing function has importance in that the user does notneed to pay attention to the progress of the assay. In emergency roomsof hospitals, for example, a nurse can add a blood sample to a deviceand insert the device into the instrument, walk away to perform otherduties and come back to the instrument, for example, when the instrumentmakes an audible sound, to get the assay results. Another example forthe utility of the timing function is that an unskilled operator of theassay system may not be able to determine when the assay has beencompleted. In this case, the timing function provided by the IAC allowsthe instrument to judge when the assay process is complete.

A preferred configuration of the timing function is to use the signalintensity of one or more IAC, for example, the flow control and/or thetime gate control, as a measure of assay completion. This embodimentfunctions when an immunoassay is started by the addition of sample to anassay device. As with all embodiments of the instant invention, thesample reconstitutes the reagents, for example, the assay reagents andthe IAC reagents in the reaction chamber, they reach substantialequilibrium binding, and the reaction mixture flows through thediagnostic lane. As the IAC reagents flow in a lateral fashion throughthe diagnostic lane, the signal intensity on the discrete zones of theIAC increases. The IAC can also be designed to have a measurable signalprior to addition of the sample. This would be accomplished by additionof a soluble label to the IAC solutions prior to or at the time ofapplication to the solid phase. When the reaction mixture flows throughthe diagnostic lane, the label in the IAC is washed out. The signal ofthe IAC would thus decrease as the label is removed by the reactionmixture occurs as a result of the assay process. Once all the reactionmixture has flowed through the diagnostic lane, the rate of increase ofthe signal of the IAC (and the analyte assays) decreases in apredictable manner.

In a particularly preferred embodiment of the timing function, the rateof change of the label bound at one or more discrete zones on the assaydevice, that is, the rate of change of the signal intensity, is ameasure of the completion of the assay process. The absolute rate ofchange of the label binding to discrete zones in the diagnostic lane fordefining the completion of the assay is arrived at empirically.

A particularly preferred location in the device for measuring the timingsignal is at the end of the diagnostic lane. This location isparticularly preferred because the end of the diagnostic lane is thelast to be washed of unbound label. Therefore, when the end of thediagnostic lane is free of unbound label, the beginning of thediagnostic lane, that is, the closest to the reaction chamber, is alsofree of unbound label.

In yet another particularly preferred embodiment of the timing function,the negative rate of change of the total label in the diagnostic lane atone or more zones of the assay device is a measure of the completion ofthe assay process. The acceptance by the timing function of a negativerate of change of the label for assay completion is related to thesensitivity requirements of the assay. That is, a high rate of change ofthe signal implies that the assay process is still taking place, butdepending on the application of the assay, the assay process may need tobe only 75% complete for an acceptable answer. Conversely, a slownegative rate of change of the signal indicates that the assay processis complete or nearly complete and this condition may be desirable whenvery sensitive and accurate results are required. The negative rate ofchange of the signal approaches zero as the washout of the labelapproaches completion. In many applications of the invention and inpractice, the negative rate of change can be a non zero number, wherethe non zero number relates to a condition whereby the majority of thelabel has been washed from the diagnostic lane. In the strictest sense,the negative rate of change becomes zero when the washout is complete.

A preferred embodiment of the timing function also measures one or moreabsolute signal intensities in one or more discrete zones as well as anegative rate of change of signal. This embodiment is important becausethe instrument must be programmed to distinguish assay devices that havehad no sample added from those that have had sample added. In the casewhen sample is not added to the assay device, the rate of change oflabel, measured by the instrument during the timing function, would bezero, and the instrument could interpret this result to mean that theassay is complete although the assay was never begun. In addition, whensample has been added to the device and the signal has reached amaximum, that is, the device has not washed the label from thediagnostic lane and the immunoassay has not been completed, then therate of change of the signal can also reach zero. Thus, the instrumentshould confirm that the sample has been added to the assay device,regardless of the specification of the rate of change of the label, byadditionally specifying the achievement of a defined signal. The definedsignal would be predictable within a range. The maximum signal in therange would be that obtained by measuring the total label and theminimum signal would be defined by the non-specific or specific bindingof the label to the device. The signal utilized for defining assaycompletion is chosen to be greater than the signal obtained when nolabel has flowed in the diagnostic lane and greater than a predeterminedvalue that would be derived from a typical non-specific binding signalwhen all the label has been washed from the diagnostic lane. Thus, thecriteria for defining assay completion in an assay device are arrived atempirically.

A complication that has been observed in measuring the signals fortriggering a measurement of the assay signals is the variation of thenon-specific binding signal in a single device. The novel teachingsdescribed herein obviate this complication. The signal to backgroundratio can potentially be high or less defined than desired at certainzones in the diagnostic lane, for example due to sample matrixvariability and imperfections in the surface of the diagnostic lane.Therefore, the analysis of an absolute signal by the timing function totrigger a measurement of the analyte assay zones should include someform of signal averaging. One skilled in the art will recognize thatthere are many methods for reducing noise in a slowly varying function.One of the simplest methods is to use an N point smoothing function,where each point is replaced with the average of N points surroundingit. Even with N point smoothing it may be likely that two consecutivepoints which meet the stability criterion is achieved before the desiredrate of change has actually occurred. The probability of thispotentially false trigger can be reduced to an acceptable level byrequiring a certain number of smoothed points in a row to meet thestability criterion.

In a preferred embodiment stability criteria include the requirement ofa negative first and positive second derivative. This will prevent apremature trigger when a local maximum is being measured rather than theminimum signal. These derivative criteria are problematic in that, ifthe device is measured by the instrument after all the signal has flowthrough the device, then both the first and second derivatives are zero,plus and minus instrument noise, so this condition become statisticallyimprobable. This adds time to reach the stability criterion. One canrecognize this condition since the signal level typically will be muchless than the high threshold. In a preferred embodiment the slopecriterion are bypassed when the signal is some fraction below the highthreshold.

An important aspect of the IAC timing function is to recognize, andalert the user, that a device does not run properly. Timeouts areincluded that require sample to flow and stability to be reached withina certain times.

In a preferred embodiment a programmable duty cycle is incorporated.This reduces power requirements, prolonging battery life.

Preferred reagents for the timing control are fluorescent particlesabout 50 nm to 300 nm in diameter and have attached an antibody, aligand or a ligand analogue. The solid phase reagent is preferably aligand, ligand analogue or an antibody, respectively. A particularlypreferred reagent for the timing control comprises the total fluorescentlabel in the assay device.

In another preferred embodiment of the invention, the IAC define theshape of an integrated signal. The definition of the shape of anintegrated signal is important in that the slope of the rise and fallfunctions of the signal defines important parameters with which theinstrument measures the analyte zones for determining the concentrationof the analyte. The shape of the integrated signals is important and candefine problems with the mechanism for measuring the signal, for examplethe optics or a means for moving an assay device by the optics, as wellas problems associated with the assay device. For example, if the meansfor binding the label to the solid phase, for example, an antibody, isapplied to the device surface in an area smaller or larger than desired,the integrated signal would have a smaller or larger value,respectively, resulting in erroneous results. The IAC would define theincorrect shape of the integrated signal of the zone and give the useran error message, defining that the assay results may not be reliable.In a system where the measurement is made over time or space one canutilize the expected behavior of the assay with the measured behavior asa method of Quality Control (QC) for the assay. Any assay that deviatessubstantially from the expected behavior, as defined by the profile ofthe signal is rejected, while an assay that matches the expectedbehavior is accepted. This novel QC step raises the reliability of theassay. In practice the behaviors which most critically depend on aproperly functioning assay must be identified. If an assay is monitoredover time, the signal response as a function of time could be measured.If the assay is measured over 1 or more dimensions, then one should lookfor characteristic shapes in area of interest. This type of analysis issimilar to a matched filter analysis.

In a preferred embodiment, the response as a function of the positionalong the diagnostic lane is measured. The characteristic shape andlocation of each zone of interest, as well as any relationships betweenzones is defined by the manufacturing process of the assay device. Themaximum rate of change expected at any given assay or IAC zone as theassay progresses in the device is also defined by the manufacturingprocess, the design of the device and the assay, so any deviation in themeasured results from the expected results is interpreted by theinstrument to be a glitch. One can filter the data with the usualassortment of high pass, low pass, and notch filters, as well as fitsections of the curve to specific functions, or use any type of exoticdigital algorithm. These types of filters remove unwanted features suchas glitches. This type of filtering is most beneficial in that itperforms a local quality control check, and helps improve the precisionof the result. One can verify that the filtering is applied only tosignals within the maximum allowable signal to background of the assayand utilize the signal to background as an internal quality controlcheck. Preferred compositions that define the shape of the fluorescentsignal are the flow control reagents, the time gate control reagents,the reaction chamber control reagents, the assay reagents orcombinations of these reagents.

In another preferred embodiment of the invention, the IAC define andverify the location of the zones of integrated fluorescence in the assaydevice. The location of the assay zones is critical for accuratemeasurement of the assay signals. For example, in the manufacture of theassay device, when the solid phase reagents for binding the label areapplied to the device in discrete zones, misplacement of the reagents onthe device could result in erroneous results. In another example, thegears on a motor that drives the assay device under an optical block formeasurement of the assay signals could slip such that the absoluteposition that the instrument reads would not be coincident with thelabel bound to the discrete zones. This scenario would also result inerroneous results. The IAC would define these potential problems and theinstrument would give an error message in response to an unexpectedlocation of an integrated signal. The error signal would notify the userthat the results should not be trusted. Preferred compositions thatdefine and verify the location of the zones of the integratedfluorescent signal are the flow control reagents, the time gate controlreagents, the reaction chamber control reagents or combinations of thesereagents.

In another preferred embodiment, the IAC redefine assay signals (of theanalytes) for purposes of correction of the assay signals as a functionof sample matrix differences, external and internal perturbations,variation in incubation times and sample flow rates in assay devices. Inthe development of immunoassays that are performed directly in viscousor heterogeneous samples, for example, as found with biological sampleslike blood, lysed blood, plasma, serum and urine, flow rates, bindingefficiencies, binding interferences and the like are encountered. Theresult of these encounters is that virtually each sample can bedifferent and the differences can result in abnormalities in the assayresults. For example, in performing an immunoassay in a lysed wholeblood sample, the viscosity of the sample can vary depending on thehematocrit of the blood sample. An increase in the viscosity of thesample causes the sample fluid to flow more slowly in a capillary of theassay device. As the flow rate decreases in the capillary and thebinding reaction occurs as the fluid flows past the binding zones, therate of binding to a solid phase can be increased because the sampleresides near the binding site for a longer period of time. This scenarioyields an assay signal that is greater than would be expected and as aresult, the calculation of the analyte concentration would be in error.In addition, as the viscosity of a sample increases, the rate ofdiffusion decreases. The decrease in the rate of diffusion increases thetime needed to approach equilibrium binding. If the immunoassay isdesigned to be at equilibrium when the instrument measures the assay,and the assay has not come to equilibrium, then the results of the assaymay be erroneous. In yet another example, if a sample contains acomponent that binds to or in some way affects the binding reactions ofthe immunoassay, such as, for example as is observed when heterophilicantibodies are present in a patient's blood, then the immunoassayprocess can be jeopardized and the assay results can be erroneous. Therole of the IAC in this embodiment would be to correct the assay signalbased on a change of the IAC. In addition, the IAC could result inreporting an error message to the user, particularly if the non-specificbinding component to the solid phase is abnormal. Preferred compositionsof reagents that redefine the assay signals for purposes of correctionof the assay signals are the non-specific binding control reagents, theflow control reagents, the time gate control reagents, the reactionchamber control reagents or combinations of these reagents.

In yet another preferred embodiment of the invention, the deviation froma mean value of the IAC is used to correct the assay in order to improvethe imprecision of assay results in different assay devices using thesame or different samples. For example, variations in hematocrit ofblood samples will affect the viscosity of the sample and therefore theflow characteristics in a lateral flow device. This embodiment relatesto the estimation of a mean signal intensity of the IAC and the extentof deviation from that mean. It is a feature of this invention to teachhow to utilize deviations from a predicted value of the IAC to reassignvalues to assay results.

In yet another preferred embodiment of the invention, the deviation froma mean value of the ratio of at least two identical IAC signals is usedto verify that a predictable immunoassay has been performed, and thatwithin a scope of high probably, the immunoassay results should bereliable.

In yet another preferred embodiment of the invention, the deviation fromthe mean value for multiple IAC are used to correct the assay in orderto improve the imprecision of assay results in different assay devicesusing the same or different samples. In a preferred embodiment thedeviation of an IAC can be influenced by the deviation of other IAC. Forexample, two IAC may be present on the device, one that is primarilysensitive to the flow rate of the device, and one that is primarilysensitive to the incubation time of the device. This embodiment teacheshow to utilize both controls simultaneously even when the results ofeach IAC is influenced by the result of the other. This teaching willrely on the principles of linear algebra.

In principle, the assay system can be perturbed by many factors, such assample heterogeneities, interfering substances and the like and it wouldbe desirable to develop an IAC for each variable that affects the assay.In this ideal case the IAC are said to be independent or orthogonal.More generally the IAC may be dependent on one another, but since theyare different it is assumed that they respond differently to variationsin different parameters, and they are therefore said to be linearlyindependent. If this assumption is not correct the controls provideidentical information. In the context of linear algebra this means thatthe controls form a basis for the parameters which influence the assay.For example, a change in flow rate in the diagnostic lane may affect thevalue of the time gate control as well as the flow control. Although thecontrols are dependent, the two in conjunction provide a uniquedescription of the flow rate and time gate time, so both controls areused to normalize the result.

A particularly preferred embodiment of normalizing the assay signals bythe IAC teaches that the source of the variability in the assay does notneed to be identified or that an IAC exist for each variable that mayarise. For example, in the description above, it was assumed that flowand time gate time are the two important variables. However, these areonly labels given to controls that may in fact be sensitive to otherparameters. To utilize the IAC in this preferred embodiment, it isassumed that there are n parameters that affect the assay results and nlinearly independent controls for these parameters. For a given sample,the mean of many assays defines the average or expected result for thecontrols (C_(ave)) and the assay (T_(ave)). In the embodiment of oneparameter, P, that affects the assay result, the measured control value(C_(m)) is equal to the expected control value plus a term proportionalto the variation of the parameter and is described as:C _(m) =C _(ave) +α δP  eqn. j1It follows that the measured value of the test can be described as:T _(m) =T _(ave) +β′ δP  eqn. j2In the analysis that follows it is assumed that α and β′ are notfunctions of δP. This is a good assumption when δP is small. α and β′can have functional dependence on the value of P. The following can beapplied for small variations around multiple values of P yielding thefunctional form with respect to P. When the variations of the parameterare small, α and β′ are constants that represent the derivatives of thecontrol and test with respect to the parameter (dC/dP and dT/dP). Theseequations can be more simply written:δC=α δP  eqn. j3δT=β′ δP  eqn. j4and combined to form:δT=(β′/α)δC=β δC  eqn. j5From this, β is found, which is easily accomplished experimentally.There is no need to ever identify or solve for the parameter.

In a preferred embodiment where there are n parameters, P, and nocontrols, C, the following similar equation is written:δC=α δP  eqn. j6Note that now the variation in the controls (δC) and the variation inthe parameters (δP) are both vectors while the partial derivatives(∂C_(k)/∂P_(j)) form an n by n matrix (α). If each control is purely afunction of one parameter then the α matrix is diagonal. If the controlsare linearly independent than an inverse for α will exist.In an analogous way it can be written:δT=β′ δP  eqn. j7Again the partial derivatives (∂T/∂P_(j)) form an n dimensional vector(β′). From linear algebra it can be written that:δP=α ⁻¹ δC  eqn. j8Using this it can also be written:δT=β′ α ⁻¹ δC=β δC  eqn. j9Note the similarity to equation j5. Again it is not necessary to eversolve for α, instead it is possible to determine β experimentally. Theinventive teachings described herein show that there is no need toidentify or solve for the parameters that are varying. Equations j5 andj9 can be used for correcting assay results as well as for determiningβ. When used to correct an assay result (T_(m)), the variation of assayresults (δT) can be expressed as the difference between the measuredassay result (T_(m)) and the corrected assay result (T_(c)). Fordetermining corrected assay results, equation j9 can be expressed as:T _(c) =T _(m) −β δC  eqn. j9a

While there exists many ways for determining β_(j) the above method doesnot influence the teachings presented above. For example, one method isto guess. While this can be time consuming, one skilled in the art willrecognize that many computer algorithms will efficiently search an nparameter space and find the best value for β_(j). When controls areindependent of each other, each β_(j) can be found from the slope of δTversus δC_(j). In a complex but more general approach, each β_(j) can befound by maximizing the correlation between control variation and assayvariation, and minimizing the standard deviation of the percent assayvariation. A straightforward and very general method is described below.Included are some important special case results that are derived fromthis approach.

The n elements of β (β_(k)) can be determined experimentally byperforming a statistically significant number of assays, which containrandom fluctuations in the IACs. The results are averaged to define theexpected test value (T_(ave)) and the expected value for each IAC(C_(kave)). For each assay the variation in the test (δT_(j)) and IACs(δC_(ki)) can readily be determined. Using equation j9 and summing overall devices, represented by i, we can write for each of the n IAC:Σ_(i) (δT _(i) δC _(ki))=Σ_(i) {Σ_(j) (β_(j) δC _(ij)) δC _(ki)}=Σ_(j)(β_(j)Σ_(i) {δC _(ij) δC _(ki)})  eqn. j10Equation 10 defines a system of n equations (for the n IACs) with nunknowns (the elements of β). To simplify the form of equation 10, wedefine a symmetric matrix, K, as follows:K _(jk)=Σ_(i) {δC _(ij) δC _(ki) }=K _(kj)  eqn. j11Using equation j11, equation j10 can be simply expressed in thefollowing two forms:Σ_(i) (δT _(i) δC _(i))=K β  eqn. j12β=K ⁻¹ Σ_(i) (T _(i) δC _(i))  eqn. j13Equation j13 defines the solution to the system of equations representedby equation j12. The method for finding the inverse of a n×n matrix canbe found in any text on linear algebra. See, e.g., Matrixes and Tensorsin Physics (2^(nd) edition), A. W. Joshi, page 47).

One skilled in the art will recognize that K_(jk) is the covariance ofthe random distributions represented by the variations in the IAC. See,e.g., Probability Distributions: An Introduction to Probability Theorywith Applications, 1972, C. P. Tsokos, page 367 for a discussion of thecovariance of two random variables. If two random variables areindependent then the covariance is zero. Therefore, for independentcontrols, K_(jk)=k_(j)δ_(jk) i.e. K is a diagonal matrix. For thisspecial case, the inversion of K is trivial and equation j13 can bewritten as follows:β_(j)=Σ_(i) (δC _(ij) δT _(i))/Σ_(i) (δC _(ij))²  eqn. j14One skilled in the art will recognize this as the equation from theleast squares fit of a line with 0 intercept. In other words, when thecontrols are independent, the slope of δT versus δC_(j) (i.e. ∂T/∂C_(j))is β_(j).

In the teaching above, the matrix (β) relating the variations in thecontrols to variations in the test (analyte assay) is found for aspecific test value, but it was not assumed that β was not a function ofthe test value. The functional dependence of β on the test value (T) canbe determined experimentally by finding β as described above atdifferent test values.

In a preferred embodiment β is found to be proportional to the magnitudeof the analyte assay, that is:β_(j)=Γ_(j)T_(ave)  eqn. j15

By using equation j15, equation j9 can be rewritten as:T _(m) =T _(ave)+Σ_(j) β_(j) δC _(j) =T _(ave) (1+Σ_(j) Γ_(j) δC _(j)),or  eqn. j16T _(ave) =T _(m)/(1+Σ_(j) Γ_(j) δC _(j))  eqn. j17Note that equations j17 and 17.5 (see example 17) are the sameequations. Equation j17 may be used to normalize assay results when oneor more IACs present (either dependent or independent) and where therelation to the control variation is proportional to the magnitude ofthe test or assay results.

In a preferred embodiment where there is only one control, equation j15holds, and Γ is equal to 1/C_(ave), equation j17 reduces to:T _(ave) =T _(m)/(1+(C _(m) −C _(ave))/C _(ave))=T _(m) C _(ave) /C_(m)  eqn. j18This is the simple case where the test (assay) value scalesproportionally with the control (IAC) value. That is, a factor of twochange in the control value will result in a factor of two change in thetest value. This is an IAC that responds identically to variations thatalso affect the assays of the analytes. One skilled in the art will nowrecognize that when an assay and an IAC change proportionately, thistype of normalization is utilized.

In the equations above, we presented an idealized case of a system withno noise. One skilled in the art will recognize that the addition of arandom noise term does not influence the methods presented above. Thisarises from the fact that random noise (δN) is by definition anindependent variable with a mean of zero. As noted above the covarianceof independent variables is also zero, so all terms involving randomnoise and other variables are zero. The only surviving term is δN².Because the random noise must be the small compared to other variations,(otherwise there is no reason to apply control correction) δN² isnegligible. In other words the techniques described above require thatvariations in the parameters which are the dominate variations in thetest be the predominate variation in the controls. Calculating thecorrelation between control variation and test variation can test forthis condition.

EXAMPLES

The following examples are embodiments of IACs of the invention. Theseexamples are for illustrative purposes only and do not limit theinvention in any manner.

Example 1 Preparation of Fluorescent Energy Transfer Latex With BovineSerum Albumin (FETL-BSA) and Antibody (FETL-AB) Conjugates

Fluorescent energy transfer latex was prepared as described in U.S.patent application Ser. No. 08/409,298, filed 23 Mar. 1995 using siliconphthalocyanine bis (di-methylhexylvinylsilyloxide) and silicon[di(1,6-diphenylnaphthalocyanine)]diphthalocyanine bis(di-methylhexylvinylsilyloxide) as donor and acceptor dyes,respectively, at a ratio of 4 moles of donor to 1 mole of acceptor. Thelatex particles are about 230 nm in diameter and were purchased fromInterfacial Dynamics Corporation. The FETL particles were adsorbed withbovine serum albumin (BSA) or various antibodies using techniques thatare standard to one skilled in the art. Alternatively, the BSA or theantibodies were attached covalently to the particles usingheterobifunctional crosslinking reagents (SMCC and SPDP, from PierceChemical Co.) also using techniques that are standard to one skilled inthe art. These methods are outlined in the Pierce Chemical Co.catalogue, 1994, p. T166 and T192, in Uniform Latex Particles, SeradynInc., p 31-40 and in Microparticle Reagent Optimization, Seradyn Inc.,p. 91-97. The unbound BSA was purified from the FETL-BSA bycentrifugation at top speed in an Eppendorf centrifuge, model 5415C. Theresulting particle was reconstituted in a buffer solution consisting of10 mM potassium phosphate, 2 mM potassium borate, 150 mM sodiumchloride, pH 7.0 at a solids concentration of about 2% (w/v).

Antibody FETL conjugates prepared in this fashion includeanti-dansyl-FETL used for the flow control IAC, the anti-troponin I,anti-CKMB, anti-myoglobin FETL, anti-human chorionic gonadotropin (HCG)FETL conjugates used in immunoassays for measuring the concentrations oftroponin I, CKMB, and myoglobin, respectively. One skilled in the artrecognizes that the specific analyte antibody pairs listed here do notrestrict the scope of this invention and that these antibody antigenpairs are used as examples of the invention. The monoclonal andpolyclonal antibodies were prepared by standard techniques, for example,as described in Antibodies, A Laboratory Manual, Ed Harlow and DavidLane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, N.Y. Therecombinant antibodies prepared and screened for the cardiac markerassays were prepared from genetic information derived from mice byvarious procedures as described in Antibody Engineering: A PracticalApproach (Borrebaeck, C., ed), 1995, Oxford University Press, Oxford; J.Immunol. 149, 3914-3920 (1992) and as described in U.S. patentapplication Ser. No. 96/05476, incorporated by reference.

Example 2 Preparation of Flow Control FETL Conjugate for the CardiacMarker Assay

To a solution of FETL-BSA, (11 ml at 1% solids, w/v, 220 nm particles),prepared according to example 1, was added with stirring, ascorbic acidand ethylenediamine tetraacetic acid to final concentrations of 20 mMand 0.1 mM, respectively. SMCC (3.8 μl of a 60 mM solution inacetonitrile) was added with stirring and the solution was incubated atroom temperature for 2 h. The reaction was quenched by addition of 0.45ml of 0.5 M solution of taurine. The solution was applied to a gelfiltration column and 23.1 ml FETL-BSA-maleimide was eluted. A solutionof decapeptide thiol (19 μl of 4.7 mM solution) was added and thesolution was incubated at room temperature for 50 min. Decapeptide thiolwas prepared by hydrolysis in as described in Example 4. The solutionwas applied to a gel filtration column and the elutedFETL-BSA-decapeptide was centrifuged at top speed in an Eppendorfcentrifuge for 20 min. The pelleted FETL conjugate was resuspended to afinal solids concentration of 2%, w/v in a phosphate buffered solution,pH 7.0.

Example 3 Preparation of Time Gate Control FETL Conjugate for theCardiac Marker Assay

To a solution of FETL-BSA, (20 ml at 1% solids, w/v, 220 nm particles),prepared according to example 1, was added with stirring, ascorbic acidand ethylenediamine tetraacetic acid to final concentrations of 20 mMand 0.1 mM, respectively. SMCC (17 μl of a 60 mM solution inacetonitrile) was added to the solution, with stirring, and incubated atroom temperature for 2 h. The reaction was quenched by addition of 0.82ml of 0.5 M solution of taurine. The solution was applied to a gelfiltration column and 16.9 ml of FETL-BSA-maleimide was eluted.Morphine-HCTL was synthesized and hydrolyzed to the corresponding thiolderivative as described in U.S. Pat. No. 5,089,391, Example 4,incorporated by reference. Morphine thiol (0.52 ml of a 16.3 mMsolution) was added and the solution was incubated at room temperaturefor 3 h. The reaction was quenched by addition of N-ethyl maleimide to afinal concentration of 2 mM. The solution was applied to a gelfiltration column and the eluted FETL-BSA-morphine was centrifuged attop speed in an Eppendorf centrifuge for 20 min. The pelleted FETLconjugate was resuspended to a final solids concentration of 4%, w/v.

Example 4 Preparation of Time Gate Control Antibody Conjugate for theCardiac Marker Assay

To 1.0 ml of anti-morphine antibody, 36E10, at 10 mg/ml in 50 mMpotassium phosphate, 10 mM potassium borate, 150 mM sodium chloride wasadded 0.022 ml of a 2 mg/ml solution of SMCC (Pierce Chemical Co.) inacetonitrile. The solution was stirred at room temperature for 3 h. Thesolution was then purified on a gel filtration column and the elutedprotein collected was 2.4 mg/ml in 3.4 ml. To this protein fractionwhich consisted of antibody-maleimide was added 0.12 ml of a decapeptidethiol (see this example for the synthesis of decapeptide thiol). Thesolution was stirred at room temperature for 3 h and was subsequentlydialyzed overnight against 50 mM potassium phosphate, 10 mM potassiumborate, 150 mM sodium chloride, 0.02% sodium azide, pH 7.0. Therecovered protein, which consisted of anti-morphine antibody decapeptideconjugate, was at 2.8 mg/ml in 3.2 ml.

Synthesis of Acetylthiopropionic Acid

To a stirred solution of 3-mercaptopropionic acid (7 ml, 0.08 moles) andimidazole (5.4 g, 0.08 moles) in tetrahydrofuran (THF, 700 ml) was addeddropwise over 15 minutes, under argon, a solution of 1-acetylimidazole(9.6 g, 0.087 moles) in THF (100 ml). The solution was allowed to stir afurther 3 hours at room temperature after which time the THF was removedin vacuo. The residue was treated with ice-cold water (18 ml) and theresulting solution acidified with ice-cold concentrated HCl (14.5 ml) topH 1.5-2. The mixture was extracted with water (2×50 ml), dried overmagnesium sulfate and evaporated. The residual crude yellow oily solidproduct (10.5 g) was recrystallized from chloroform-hexane to afford 4.8g (41% yield) acetylthiopropionic acid as a white solid with a meltingpoint of 44-45° C.

Decapeptide Thiol Synthesis

The decapeptide thiolpropionamide (decapeptide thiol) was synthesized byhydrolysis of the decapeptide acetylthiopropionamide (decapeptide ATP).Decapeptide ATP was synthesized from the decapeptide, YPYDVPDYAS,(Chiron Mimotopes Peptide Systems, San Diego, Calif.) andacetylthiopropionic acid. Thus, the decapeptide was dissolved (0.3 g) indry DMF (5.4 mL) in a round bottom flask under argon with moderatestirring. Imidazole (0.02 g) was added to the stirring solution.Separately, acetylthiopropionic acid (0.041 g) was dissolved in 0.55 mLof dry DMF in a round bottom flask with stirring and 0.056 g of1,1′-carbonyldiimidazole (Aldrich Chemical Co., Milwaukee, Wis.) wasadded to the stirring solution. The flask was sealed under argon andstirred for at least 30 minutes at room temperature. This solution wasadded to the decapeptide solution and the reaction mixture was stirredfor at least six hours at room temperature before the solvent wasremoved in vacuo. The residue in the flask was triturated twice using 10mL of diethyl ether each time and the ether was decanted. Methylenechloride (20 mL) was added to the residue in the flask and the solid wasscraped from the flask and filtered using a fine fritted Buchner funnel.The solid was washed with an additional 20 mL of methylene chloride andthe Buchner funnel was dried under vacuum.

The thioacetyl moiety of the decapeptide ATP was hydrolyzed bydissolution in 70% DMF to make a 20 mM solution. A solution of 1 Npotassium hydroxide was added to a final concentration of 0.2 N whilemixing vigorously. The solution was incubated at room temperature for 5minutes prior to neutralization to pH 7 of the solution by the additionof a solution containing 0.5 M potassium phosphate, 0.1 M borate, 1 Mhydrochloric acid. The thiol concentration of the decapeptide thiolderivative was determined by diluting 10 μL of the solution into 990 μLof a solution containing 0.25 mM 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB, Aldrich Chemical Co., Milwaukee Wis.) and 0.2 M potassium borate,pH 8.0. The thiol concentration in mM units was equal to theA₄₁₂(100/13.76).

Example 5 Preparation of Solid Phase Antibody, Avidin and Dansyl LatexConjugates

The solid phase antibody latexes were prepared by adsorbing antibodiesor bovine serum albumin to particles of about 130 nm in diameter.Procedures for adsorption of these proteins to latex particles arefamiliar to one skilled in the art and can be found in Uniform LatexParticles, Seradyn Inc., p 31-32 and in Microparticle ReagentOptimization, Seradyn Inc., p. 91-97.

The avidin latex, which was used as the IAC for the cyclosporin assay,was prepared by adsorption of avidin (Pierce Chemical Co) to latexparticles as described in the references cited above.

The dansyl latex, which was used as the flow control solid phase for thecardiac assay, was prepared by reacting the bovine serum albumin latexwith SMCC according to example 3. The resulting latex BSA-maleimide wasreacted with dansyl thiol. The dansyl thiol was prepared by reduction ofdidansylcystein (Molecular Probes) with tributyl phosphine (AldrichChemical Co). The resulting dansyl BSA latex conjugate was purified on agel filtration column and the eluted solids was centrifuged at top speedfor 20 min in an Eppendorf centrifuge.

The solid phase latex solutions were reconstituted in phosphate buffer,pH 7.0 at a solids concentration of 1%, w/v.

The slid phases prepared in this manner were utilized for the time gatecontrol of the cardiac assay, which comprised an anti-decapeptideantibody conjugate latex, for the flow control of the cardiac assay, adansyl BSA conjugate latex, for the non-specific binding control of thecardiac assay, an anti-benzodiazapine antibody conjugate latex, for thetroponin I assay, an anti-troponin antibody conjugate latex, for theCKMB assay, an anti-CKMB antibody conjugate latex and for the myoglobinassay, an anti-myoglobin conjugate latex. The solid phases utilized forthe cyclosporin assay comprised an avidin conjugate latex for the timegate and flow controls and an anti-phencyclidine antibody conjugatelatex for the cyclosporin assay.

Example 6 Preparation of FETL-Cyclosporin

FETL-BSA, (25 ml at 1% solids, w/v, 220 nm particles), preparedaccording to example 1, was reacted with 0.22 mgcyclosporin-N-hydroxysuccinimide (Novartis, Basel, Switzerland) afterthe addition of 2.5 ml dimethylformamide. The reaction stirred at roomtemperature for 30 min after addition ofcyclosporin-H-hydroxysuccinimide. The reaction was quenched by additionof 1.1 ml of 0.5 M solution of taurine. The mixture was applied to a gelfiltration column. The eluate containing the FETL-cyclosporin wascentrifuged at top speed in an Eppendorf centrifuge for 20 min. Thepellet was reconstituted in 10.5 ml 10 mM potassium phosphate, 2 mMpotassium borate, 150 mM sodium chloride, pH 7.0 to a solidsconcentration of 2.2%, w/v.

Example 7 Preparation of FETL-Decapeptide Conjugate (IAC Label forCyclosporin Assay)

To a solution of FETL-BSA, (15 ml at 1% solids, w/v, 220 nm particles),prepared according to example 1, was added with stirring, SMCC (25 μl ofa 2 mg/ml solution in acetonitrile). The solution was incubated at roomtemperature for 2 h. The reaction was quenched by addition of 0.6 ml of0.5 M solution of taurine. The solution was applied to a gel filtrationcolumn and 23.1 ml FETL-BSA-maleimide was eluted. A solution ofdecapeptide thiol (19 μl of 4.7 mM solution, see example 4 for thesynthesis of decapeptide thiol) was added and the solution was incubatedat room temperature for 50 min. The solution was applied to a gelfiltration column and the eluted FETL-BSA-decapeptide was centrifuged attop speed in an Eppendorf centrifuge for 20 min. The pelleted FETLconjugate was resuspended to a final solids concentration of 2%, w/v ina phosphate buffered solution, pH 7.0.

Example 8 Preparation of Anti-Cyclosporin Phencyclidine Antibody andAnti-Decapeptide Biotin Antibody Conjugates

Monoclonal antibodies to cyclosporin and decapeptide were used for theassay of cyclosporin and the IAC of the cyclosporin assay, respectively.These antibody conjugates were placed on the lid of the assay device andare termed lid antibodies. The anti cyclosporin antibody was reactedwith phencyclidine thiol and the anti decapeptide antibody was reactedwith biotin.

Thus, to a solution of anti cyclosporin antibody (1.0 ml at 10 mg/ml,Novartis) in 50 mM potassium phosphate, 10 mM potassium borate, 150 mMsodium chloride, pH 7, was added 22 μl of 2 mg/ml SMCC in acetonitrilewith stirring. The solution was stirred at room temperature for 3 h. Thesolution was added to a gel filtration column equilibrated in 50 mMpotassium phosphate, 10 mM potassium borate, 150 mM sodium chloride, pH7. The anti cyclosporin maleimide was collected and was reacted withphencyclidine thiol. The phencyclidine thiol was prepared by hydrolysisof phencyclidine ATP. Phencyclidine ATP and phencyclidine thiol weresynthesized according to examples 4 and 5, respectively, in U.S. Pat.No. 5,331,109. A solution of phencyclidine thiol (90 μl of 18 mM) wasadded to the anti cyclosporin maleimide with stirring. The solution wasstirred at room temperature for 2.5 h. The antibody phencyclidine wasapplied to a gel filtration column equilibrated in 50 mM potassiumphosphate, 10 mM potassium borate, 150 mM sodium chloride, pH 7. Theeluted protein was collected in a 4 ml aliquot at 1.2 mg/ml.

To a solution of anti decapeptide antibody (0.5 ml at 10 mg/ml) wasadded 0.1 ml of 2 mg/ml biotin-N hydroxysuccinimide (Pierce ChemicalCo.). The reaction stirred at room temperature for 2 h. The proteinsolution was added to a gel filtration column equilibrated in 50 mMpotassium phosphate, 10 mM potassium borate, 150 mM sodium chloride, pH7. The anti decapeptide biotin conjugate was eluted in 1 ml at 4.2mg/ml.

Example 9 Preparation of Independent Assay Control for CyclosporinAssays

The IAC for the cyclosporin assay comprised an FETL decapeptideconjugate (example 7) which was added to the base of the assay device inthe reaction chamber with the FETL cyclosporin conjugate, an antidecapeptide biotin conjugate (example 8) which was added to the lid ofthe assay device with the anti cyclosporin biotin conjugate and anavidin conjugate latex (example 5) which was applied to the solid phaseof the device in the diagnostic lane.

The solids concentration of the FETL decapeptide conjugate and the FETLcyclosporin conjugate was 0.04%, w/v in the reaction mixture. Theantibody concentration of the anti decapeptide biotin antibody conjugateand the anti cyclosporin biotin antibody conjugate was 0.3 μg/ml in thereaction mixture. One skilled in the art will recognize that theabsolute signal of the IAC can be adjusted higher or lower by raising orlowering, respectively, the concentrations of the IAC FETL and antibodyconjugates, separately or together, in the reaction mixture.

Example 10 Preparation and Description of Assay Devices

Assay devices as described in U.S. Pat. No. 5,458,852 are utilized forthis example; however, one skilled in the art recognizes that many assaydevices and formats can be utilized for application of reagents andpracticing the inventive teachings described herein. For example, assaydevices incorporating membranes, as described in U.S. Pat. Nos.4,200,690, 4,391,904, 4,435,504, 4,857,453, 4,963,468, 5,073,484,5,096,837 and 5,654,162, incorporated by reference only, also can beadapted to utilize the inventive teachings described herein.

Compositions of FETL-antibodies and FETL-ligands are added to thereaction chamber of the base so that the final assay solidsconcentration is between about 0.2% and 0.4% (w/v). The time gatecontrol antibody is applied to the lid in the area of the reactionchamber to a final assay concentration of about 5 μg/ml. The solid phaseantibody latexes are applied to the diagnostic lane in zones of about 2mm×3 mm. In the case of assays utilizing a filter to remove red bloodcells from plasma and debris in general from the sample, the device andfilter are configured as described in U.S. patent application Ser. No.08/704,804, filed Aug. 26, 1996, incorporated by reference. In the caseof assays incorporating a lysis mesh for lysing whole blood, the deviceand lysis mesh are configured as described in U.S. Pat. No. 6,106,779,incorporated herein by reference in its entirety, including all figures,tables, and drawings. The time gate is applied to the device andfunctions as described in U.S. Pat. No. 5,458,852. The assay device lidis attached to the base by ultrasonic welding, as one skilled in the artwill recognize.

One skilled in the art will also recognize that filters, lysis chambers,reaction chambers, time gates and diagnostic lanes in capillary devicesare not prerequisites for practicing this invention. In addition, areaction mixture can be formed in a test tube, incubated and applied toa membrane or a capillary such that the flow of the reaction mixturewithin the membrane or capillary is in a lateral fashion. The bindingreactions of the label in the reaction mixture and the reagents on thesolid phase of the membrane or the capillary take place after additionof the reaction mixture to the solid phase. The washing of unbound labelfrom the binding zones can be accomplished by addition of a buffercontaining detergent.

In general, assay devices used in these examples each comprise a timegate control and a flow control. In some cases, the time gate controlIAC and the flow control IAC can be performed by one set of reagents.That is, a time gate control can also function as a flow control.

Immunoassay devices for cardiac markers, as described herein, eachcomprise a separate time gate control and a flow control and these IACare independent of each other. The IAC antibody for the time gatecontrol (anti morphine antibody decapeptide conjugate) is applied to thelid at concentrations that are adjusted relative to the dynamic range ofthe assay and range from about 0.1 μg/ml to 50 μg/ml and preferably 5μg/ml in the reaction mixture. The labeled conjugates are applied to thereaction chamber and the solid phase capture reagents are applied to thediagnostic lane of the assay device. The concentrations of the IAC FETLconjugates were 0.06% solids, w/v, for the flow control and 0.12%, w/v,for the time gate control. One skilled in the art will recognize thatthe absolute signal of the IAC can be adjusted higher or lower byraising or lowering, respectively, the concentrations of the IAC FETLand antibody conjugates, separately or together, in the reactionmixture.

During the assay process when sample reconstitutes the reagents in thereaction chamber to form a reaction mixture, the anti-morphine antibodydecapeptide conjugate binds to the FETL morphine conjugate. The timegate allows for an incubation, after which the reaction mixture flowsthrough the diagnostic lane, during which the time gate control antibodybinds to the time gate control label and analytes bind to theirrespective FETL antibody conjugates. The labels in the reaction mixturebind to the solid phase in the respective zones. Thus, the FETL morphineantimorphine antibody decapeptide conjugate complex binds to theimmobilized anti decapeptide antibody, which defines the time gatecontrol IAC, the FETL dansyl conjugate binds to the immobilized antidansyl antibody, which defines a flow control IAC, the total FETLconjugates bind to the immobilized anti benzodiazapine, which definesnon-specific binding to an antibody zone, the FETL anti CKMB antibodyconjugate binds to the immobilized anti CKMB antibody in the presence ofCKMB, the FETL anti troponin I antibody conjugate binds to theimmobilized anti troponin I antibody in the presence of troponin I andthe FETL anti myoglobin antibody conjugate binds to the immobilized antimyoglobin antibody in the presence of myoglobin. The IAC function tocommunicate with the instrument concerning the status of completion ofthe immunoassay and relevant information concerning non-specific bindingto the solid phase in the diagnostic lane.

Immunoassay devices for cyclosporin comprise an IAC that functionssimilarly to the competitive immunoassay format which is performed forthe measurement of cyclosporin. The cyclosporin assay device can alsocomprise a separate flow control, as described for the cardiac markerdevice. The IAC antibody (anti-decapeptide biotin) is applied to the lidwith the anti-cyclosporin antibody at concentrations that are adjustedrelative to the dynamic range of the assay. The labeled conjugates areapplied to the reaction chamber and the solid phase capture reagents areapplied to the diagnostic lane of the assay device. During the assayprocess when sample reconstitutes the reagents in the reaction chamberto form a reaction mixture, both anti-cyclosporin PCP conjugate andanti-decapeptide biotin conjugate bind to the FETL cyclosporin conjugateand FETL decapeptide conjugate, respectively. A ligand, for example inthis case, decapeptide can also be added to the mesh in the lysischamber so that it is reconstituted with the sample prior to the sampleentering the reaction chamber. The ligand functions as an analyte sothat the IAC behaves like the competitive immunoassay for cyclosporin.The time gate allows for an incubation, after which the reaction mixtureflows through the diagnostic lane. The FETL cyclosporin anti cyclosporinantibody phencyclidine conjugate binds to the immobilized antiphencyclidine antibody, which defines the cyclosporin assay and the FETLdecapeptide anti decapeptide biotin conjugate binds to the immobilizedavidin, which defines the IAC. The IAC function to communicate with theinstrument concerning the status of completion of the immunoassay andrelevant information concerning non-specific binding to the solid phasein the diagnostic lane.

Example 11 Performing an Immunoassay

Several drops of sample (about 70 μl for whole blood into a device forlysing the sample, for example in the cyclosporin assay and about 180 μlwhole blood or plasma for the device incorporating a filter) are addedto assay devices, assembled as described in Example 6. The assay devicesare placed in the instrument, immediately or any time up to about 30 minafter sample addition. See, e.g., U.S. patent application Ser. No.09/003,090, entitled “Immunoassay Fluorometer,” Buechler et al., filedJan. 5, 1998, now U.S. Pat. No. 6,830,731, and U.S. patent applicationSer. No. 09/003,066, entitled “Media Carrier for an Assay Device,”Buechler et al., filed Jan. 5, 1998, now U.S. Pat. No. 6,074,616.Several simple commands are prompted by the instrument, which oneskilled in the art would be capable of performing and the instrumentmoves the assay device under the optical block for analysis of thecapture zones and zones utilized for the timing function. After theinstrument determines that the immunoassay in the device is completed, aresult appears on the screen showing the concentration of the assayedanalytes.

Example 12 Method for Defining Specific Capture Zone Locations in aDiagnostic Lane Using the IAC

The diagnostic lane can be separated into three types of regions, thebaseline zones, the capture zones, and the capture zone edges. FIG. 10illustrates these regions. The identification of these regions willallow a different filter to be applied to each region, as described inexample 13. The size and location of each of these regions will beutilized as a method of validating the trace, as described in example14.

Two spots with known high signal to noise ratio are used as locatingzones. In this assay these are two controls zones, as shown in FIG. 10.FIG. 10 represents the fluorescent scan of a cardiac panel device onwhich blood spiked with CKMB, troponin I and myoglobin was assayed. Ifthe edges of each locating zone can be found and there is proper spacingbetween locating zones, then the expected location of the other zones isdetermined since the spacing between all the zones is a constant that isfixed by the solid phase application process. Therefore, the edges ofthe locating zones are used to define the expected locations of all theother zones. This procedure removes any positioning uncertainties by themeasurement system or by the solid phase application system.

The capture zone edges are identified by their sharp rise out of thebackground signal. The approximate average signal height of the zone isutilized to define threshold heights that are 25, 50, and 75% of theaverage signal height. See FIG. 11. The point where the signal crossesthe threshold defines the location of the threshold crossing. Oneskilled in the art will recognize that for a 50% threshold the left andright hand crossings define the full width at half maximum. This iscommonly used as the width of the curve. The presence of noise can causean error in the determination of the threshold crossing. To minimize theeffect of noise on the determinations, the location of each thresholdcrossing is found by searching the edge in both directions, high to lowand low to high. If the location of the threshold crossing isindependent of the search direction then it has high reliability and ispreferred over crossings that are dependent on the search direction. Fora well-defined edge that is not affected by noise, the location of eachthreshold crossing is independent of the search direction and is withinthe expected range of positions of the transition. Reliability of thethreshold crossing is rank by the following criteria.

1) The threshold crossing does not exist.

2) The threshold crossing exists.

3) The threshold crossing is direction independent.

4) The threshold crossing is within the expected range of positions ofthe transition.

5) The threshold crossing is within the expected range of positions ofthe transition and is direction independent.

The threshold crossing with the highest reliability defines the qualityof the edge. The quality of the capture zone is defined by the edge withthe poorest quality.

The location of the threshold crossing with the highest reliability isused to define a point on the capture zone edge, and the remainder ofthe capture zone edge is defined as the monotonic region around thispoint. Because noise in the signal can prematurely end the monotonicregion, one non-monotonic point is allowed to be included in the capturezone edge provided that the next two points are again monotonic.

When the edges of each capture zone have thus been found, the trace canbe divided into the three types of regions. Each of these regions willbe treated differently by the custom digital filter. By definition theedges are rapidly changing monotonic regions. There is no need to filterthese regions, so they are left unchanged. Example 13 details thefiltering applied to the baseline zones and capture zones.

In FIG. 10, the capture zones (CZ) are the regions where signal isexpected. In this example, the first two capture zones have well definedsignals. Theses zones are control zones, and are used as locating zones.Capture zones on this device are the same width and equally spaced. Thebaseline zones (BL) are the regions between the capture zone. Thecapture zone edge is the transition from the baseline signal to theelevated capture zone signal.

In FIG. 11, the approximate 75%, 50%, and 25% thresholds are shown forsix zones. The heights of these zones are scaled so they can all beplotted on the same graph. The third capture zone is noisy and thelocations of all the threshold crossings depend on the directionsearched. Furthermore, the 25% threshold will not be found on theleft-hand side of the capture zone. The fourth capture zone has a spikeon the right-hand edge. The 25% and 50% crossings will be dependent onthe direction searched. The other capture zones have well defined edgesand the locations of all threshold crossings are independent of thedirection searched.

Example 13 Method for Smoothing the Fluorescence Measurement of theDiagnostic Lane and Defining the Capture Zone Signal Using the IAC

This example illustrates the use of filter algorithms in conjunctionwith the three types of zones discussed in example 12. Each zone hasunique characteristics, so the filtering applied to each zone isdistinct. As noted in example 12, the capture zone edges are rapidlychanging monotonic regions, so no filtering is applied to these zones.

The baseline zones are expected to be flat, so a low pass filter isapplied to these regions. Normally a filter acts on a signal as afunction of time, but the fluorescence signal is a function of positionalong the diagnostic lane. The low pass filter can be applied in bothdirections along the diagnostic lane. Using this symmetry each zone wasfiltered in both directions and the two results combined. Thiscombination can take many forms including maximum, minimum, and average.The combined low pass filter results form the filtered baseline. It isobserved that noise in the signal is dominated by spikes on thebaseline. Therefore, it is assumed that the minima observed within thebaseline zone represent the baseline. This dictates the use of theminimum of the left and right handed filters for the combined filter.FIG. 12 illustrates the results of this bi-directional filtering. Thefilter can also have separate time constants for increasing anddecreasing signals. Selecting (down=0) will allow the filter to returnto the baseline quickly. FIG. 13 illustrates the effects of differenttime constants. The filter can also include a maximum allowed rate ofchange. This limits the response to large glitches resulting inincreased attenuation of glitches. FIG. 15 illustrates this effect.

The capture zone signal has a characteristic shape, which is fit to afunction that approximates this shape. A parabola is a closeapproximation to the true, but unknown functional form of the capturezone. The parabola is found by a least squares fit. FIG. 14 showsexamples of some characteristic capture zones and the resultingparabolic fit.

The base line zones surrounding the capture zone are used to define thebaseline signal in the capture zone. This is accomplished via linearinterpolation. The difference between the capture zone signal and thebaseline is the signal from specific binding. This signal is summed overthe capture zone and is the capture zone result. For details concerningcapture zone derivations, see, e.g., U.S. application Ser. No.08/065,528, Buechler, “Diagnostic Devices and Apparatus for theControlled Movement of Reagents without Membranes,” filed May 19, 1993;U.S. application Ser. No. 08/828,041, Buechler, “Diagnostic Devices andApparatus for the Controlled Movement of Reagents without Membranes,”filed Mar. 27, 1997; and U.S. application Ser. No. 08/902,775, Buechler,“Diagnostic Devices and Apparatus for the Controlled Movement ofReagents without Membranes,” filed Jul. 30, 1997; all of which areincorporated herein by reference including all figures, tables, anddrawings.

The RMS deviations between the original signal and the filtered signalsare used to define the noise in both the baseline zones and the capturezones. The signal to noise of a zone is defined as the ratio of thecapture zone result to the noise of the zone.

One skilled in the art will recognize that glitches or other featuresthat can be distinguished from the signals of interest can be eliminatedprior to filtering. One method of finding these features is to usecriteria such as width, and aspect ratio to define the range of allowedshapes, then check for the shapes by performing a correlation withcandidates in the trace. Another method is to search for characteristicsignatures in the derivatives of the trace. As shown in FIG. 15, thesepre-filters can effectively eliminate a characteristic source of noise.

In FIG. 12, a section of a trace is plotted three times, each with adifferent filter applied. The LH Filter is the result when a filter isapplied moving left to right. The RH Filter is the result when a filteris applied moving right to left. The filter applied is a low pass filterwhen the signal is increasing (up=1 mm) and does not filter decreasingsignals (down=0 mm). The Combined trace is the minimum of the RH and LHfilters.

In FIG. 13, a section of a trace is plotted twice with different filtersapplied. The first filter shows the effect of (down=0, up=1 mm). Thesecond filter shows the effect of (up=down=1 mm). A predominate sourceof noise is spikes on top of a baseline signal. The use of theasymmetric filter preferentially selects for the lowest points.

In FIG. 14, characteristic shapes of capture zones with theircorresponding parabolic fits are shown. The heights of these zones arescaled so they can all be plotted on the same graph.

In FIG. 15, the example shows a glitch with three different filtersapplied. The first filter is the combined filter of FIG. 12, with nolimit on rate of change allowed. The second filter is also the combinedfilter, but now there is a maximum allowed rate of change. The thirdtrace shows the effect of a glitch filter.

Example 14 Methods for Rejecting Assay Data Based on Faulty Devices orSamples Using the IAC

The trace QC algorithm must decide if the assay results are valid.Validating each capture zone is accomplished by utilizing severalcriteria, including capture zone width and location, capture zone edgewidth and quality, and capture zone S/N and baseline S/N. While it iseasy to define what constitutes acceptable and unacceptable criteria,there often remains a gray area between the acceptation and rejectioncutoffs. A series of complex rules or fuzzy logic is required to decidethe fate of a questionable assay. The digital filter treated capturezones differently than the baseline. This filter relied on an accuratedetermination of the capture zone edge. Therefore, if a capture zonefails the trace QC, it is assumed that the edges were incorrectlyidentified and the capture zone is filtered the same as the baselinezones.

The trace is validated by first rejecting bad capture zones, thenaccepting good capture zones, and then resolving any questionablecapture zones. These three steps are performed in the following manner.The width of the capture zone, the location of the capture zone, the S/Nof the capture zone, and the S/N the baseline zones are all compared torejection criteria. If any of these four tests fail the capture zone isunacceptable, i.e. it failed trace QC. For remaining capture zones, theS/N of the capture zone and the edge quality are compared to acceptancecriteria. A capture zone passes trace QC if it passes these two tests. Aspot is questionable if it passes the rejection criteria but fails theacceptance criteria. Secondary criteria are used to resolve questionablecapture zones. The secondary criteria require that both the capture zonewidth and the capture zone edge widths be within acceptable limits. Thecapture zone edge width is important because when the signal is small, aspike is often erroneously identified as an edge. The width of this edgeis typically narrower than the width of a true capture zone edge.

A capture zone that fails trace QC does not exhibit the qualitiesexpected from a clean signal, and therefore must be treated the same asbaseline zones. The edges of the rejected capture zone are ignored andthe baseline filter is applied to the capture zone and the twosurrounding baseline zones. This newly filtered signal is re-integrated,yielding a new capture zone result. Any result above a noise threshold,set by the system noise, should have yielded an acceptable capture zone.A result from a capture zone that failed trace QC, which is above thenoise threshold, can not be explained and must be rejected. Thus thecapture zone fails quality control and the result from the zone is notreported. If the result from a capture zone that failed trace QC isbelow the noise threshold, it is assumed signal is limited by systemnoise, and the result is reported. FIG. 16 illustrates rejected zonesthat have been filtered by the baseline filter. One capture zone isabove and one is below the noise threshold.

The baseline zones are an important part of the device quality control.Insufficient sample volume will result in a high or sloping baseline.Furthermore, as described in example 12, the baseline is subtracted fromthe capture zone signal prior to calculating the result. Therefore,accurate results require that the baseline signal behave as expected.

The baseline is validated by checking that the level is below athreshold level, or small compared to the amplitude of the adjacentcapture zones. It is also validated by checking that the slope of thebaseline zones is below a threshold level or small compared to theamplitude of the adjacent capture zones. The final validation check isthat the second derivative (kinks) of the baseline zone is below athreshold level or small compared to the amplitude of the adjacentcapture zones. FIG. 17 illustrates these tests. The amplitude (BL₀),slope (BL₁) and second derivative (BL₂) of the baseline at the capturezone are calculated. Each of these values is compared to the sum of thecorresponding threshold level (Th_(x)) and the slope value (mx) timesthe capture zone result (CZR) as shown in equation 14.1.Bl _(x) >Th _(x) +m _(x) CZR  Eqn. 14.1

The threshold level, set by the system noise, represents the maximumacceptable value when there is no capture zone signal. The slope value(m_(x)) sets the acceptable S/N for large capture zone signals. If abaseline zone fails quality control, i.e. equation 14.1 is true for anyx, then the results from the corresponding capture zone is not reported.Some of the capture zones are control zones and control zone resultsmust fall within an acceptable range. Each control zone result iscompared to its minimum and maximum allowed values. If any control zoneresult falls outside of the acceptable range the entire test isrejected.

In FIG. 16, two capture zones are illustrated. The first zone is noisyand does not have well defined edges. The second zone is very wide, soits edges did not fall within the allowed region. These edges of thesezones could not be accepted by the trace QC algorithm. Without edges,these zones are filtered the same as the baseline. The wide zone has aresult that is above the noise threshold so the zone result is notreported. The noisy spot has a result that falls below the noisethreshold, and is not rejected.

In FIG. 17, three traces are plotted, each with baseline problems. Goodbaselines are around 500 and are flat (see FIG. 10). The High Baselinetrace is above 10,000, and exhibits a slope problem. The SlopingBaseline is higher than desired, but within acceptable limits. However,the slope makes it difficult to resolve the small signals, and theseresults are rejected. The High 2nd Derivative signal as a distinct kink.The result from a small signal near this kink is not reliable andtherefore rejected.

Example 15 Methods for Detecting the Completion of an Immunoassay UsingIACs

In this example several methods using IACs are demonstrated for thedetection of immunoassay completion by an instrument. A preferred methodis illustrated with a spike and recovery experiment using severalimmunoassay devices and an instrument controlled by a timing algorithm.All of the schemes discussed can be used to detect assay completionregardless of when the immunoassay device is inserted in the instrument.In most cases, assay completion is defined to be when the assay signalshave reached steady state. In some cases (see Example 16), assaycompletion can occur before the assay signals have reached steady state.Steady state is defined as an absolute value of rate of change of signalthat is less than a defined limit. In a preferred embodiment, steadystate of a timing signal is defined as a negative rate of change whoseabsolute value is less than a defined limit.

Time Profiles of Assay and IAC Signals

The time-profiles of fluorescence signals from assay and IAC detectionzones on the diagnostic lane of assay devices were determined in orderto define the criteria used in timing algorithms to detect assaycompletion. The timing algorithm is a set of software instructionsutilizing mathematical functions to define the completion of the assay.Immunoassay devices (example 10) were configured with detection zonesfor CKMB, troponin I, Myoglobin, a flow control, a timegate control anda non-specific binding control (cardiac panel devices). Humanheparinized whole blood spiked with CKMB (Genzyme; 10 ng/ml), troponin I(Bio-tech International, Seattle, Wash.; 4 ng/ml) and myoglobin (ScrippsLaboratories, San Diego, Calif.; 40 ng/ml) was added (example 11) to acardiac panel device, and the device was inserted into an instrumentwithin 1 minute. The instrument was programmed to scan the diagnosticlane (measure fluorescence as a function of position on the lane) everyminute. The resultant data were transferred to a computer for analysis.The assay and control results were quantified by integrating thefluorescence over the detection zones with the background signal(measured between detection zones) subtracted (examples 12-14). Theresultant integrated signals from the CKMB, troponin I and myoglobindetection zones were used to calculate concentrations of theserespective analytes from previously determined calibration curves. Inaddition, the fluorescence signal (the timing signal) from a zone (thetiming zone) downstream of the last detection zone was quantified. Asthere were no binding proteins immobilized in the timing zone, thetiming signal was the sum of the fluorescence from unbound (unwashed)FETLs and non-specifically bound FETLS.

The time profiles of the integrated assay signals, control signals andthe timing signal are shown in FIGS. 1 through 7. In general, the timeprofiles indicate that all the signals reach steady-state at about thesame time (between 10 and 15 minutes). The data of FIG. 4 are used belowto illustrate several embodiments of the use of IACs to determine assaycompletion.

Assay Completion Detected by Measurement of IAC Signal Values

In this embodiment, assay completion is defined to occur when some orall of the IAC signals are within defined acceptance windows. In manycases, the acceptance window will bracket the steady state values of theIAC signals. For example, in the case illustrated in FIGS. 1 through 7,the acceptance windows could be defined as follows: integrated flowcontrol signal between 26 and 27, integrated timegate control signalbetween 340 and 350 and timing signal between 1 and 3. The only time allof the IACs were within these acceptance windows were after the assays(CKMB, troponin I and myoglobin) were at steady state. Therefore, themethod of acceptance windows can be used to detect steady state (assaycompletion) with only a single measurement (at only one time) of theassay device as long as the assay signals reach steady state at the sametime or before the IAC signals do. A device in which the immunoassay iscomplete when it is inserted into the instrument can be both checked forassay completion and read with only a single scan of the diagnosticlane. Therefore, measurement of said device is done as quickly aspossible after insertion into the instrument. The disadvantage of themethod of acceptance windows is that the windows must be wide enough toaccommodate the normal variation (standard deviation) in signals amongdifferent devices and/or different blood samples. The acceptance windowmust be several (more than 2) standard deviations wide for at least 95%of the devices to eventually satisfy the acceptance criteria. Too wideof an acceptance window can lead to premature measurement of the assaysignals (before steady state is reached). Therefore, this method ispractical when the coefficients of variation (C.V.) of the IAC signalsare small enough or when the variations (C.V.s) in IAC signals are wellcorrelated with the variations in the assay signals and when the controlsignals are used to normalize the assay signals (Examples 16, 18, 19, 20and 21). The method of acceptance windows can also be applied to casesin which assay completion is defined to occur before the IAC and assaysignals reach steady state, for example when assay completion is definedto occur at a fixed time in the time-profile of the assay signal. Whenthe assay is to be read before steady state is reached, and when the IACand assay signals are correlated in time, the method of acceptancewindows could lead to a better precision in the assay results than couldbe obtained by reading the assay signal at a fixed time. The betterprecision could result if the time-profiles of the assay and IAC signalswere correlated with each other but varied among different assaydevices.

Assay Completion Detected by Determination of Rate of Change of IACSignals

In this embodiment some or all of the control, assay and/or timingsignals are measured at least twice to determine if steady state hasbeen reached within a defined limit for the maximum allowed rate ofchange of the signal. The data of FIG. 4 show that the assays are atsteady state when the controls and timing signal are at steady state.Assay completion is defined to be when some or all of the control, assayand/or timing signals are at steady state. For many assayconfigurations, including that of the assay device described in thisexample, measurement of the negative rate of change of the timing signalprovides an accurate and precise means of detection of assay completion.Measurement of the timing signal alone has the advantage that theinstrument does not need to scan the entire diagnostic lane during thetiming cycle, thus saving on power consumption and mechanical wear ofthe instrument. For some assay configurations, the timing signal alonemay not provide adequate information or be large enough to determine ifthe assays have reached steady state. In these cases, in addition tomeasuring the timing signal, determination of the rate of change of someor all of the control and/or assay signals may be necessary to obtainaccurate detection of assay completion.

Use of the Timing Signal to Detect Assay Completion of ImmunoassayDevices

To illustrate the use of the timing signal to determine assay completion(steady state), six cardiac panel devices were run with human wholeblood spiked with CKMB (8 ng/ml), troponin I (1.3 ng/ml) and myoglobin(110 ng/ml). The devices were inserted into instruments within oneminute of adding the blood and the instrument software was started(example 11), placing all further control of the assay measurement underthe instrument software.

The instrument positioned the assay devices such that the timing signalzone was under the optical block in position to be measured. Theinstrument measured the timing signal every ten seconds (each timingsignal measurement is called a time-point). To smooth the timing signaltime-profile (reduce the influence of random noise), the timingalgorithm software averaged all the time-points in a window consistingof three consecutive time-points to obtain a time-point average. Byadvancing the window one time-point at a time, a series of overlappingtime-point averages was computed. Steady state was defined to be whenthree consecutive time-point averages agreed with one another to within3%. One skilled in the art will recognize that this criterion for steadystate can be met at three distinct places (times) in the time-profile ofthe timing signal (FIG. 7). The first time the criterion can be met isimmediately after the device is inserted into the meter before thetimegate has broken when there is no fluorescence in the timing zone.The second time the criterion can be met is when the timing signal hasreached its maximum value and is transitioning from rising to falling.The third time the criterion is met is when the timing signal comprisesa negative rate of change and has dropped to its final steady statevalue. Only at this third time are the assays at steady state and readyto be measured. In the best mode, in order to avoid a premature readingof the device at the first two times, the timing algorithm requiredseveral additional criteria to be met before the assay device was read.They are: (i) the value of the timing signal had to be above the levelexpected for a device with no fluorescent label in the timing zone butbelow the maximum value expected in the timing signal time-profile (theacceptance window was a timing signal between 0.7 and 200 timing signalfluorescence units (FIG. 7); (ii) the first derivative of the timingsignal had to be negative and (iii) the second derivative of the timingsignal had to be positive. One skilled in the art will recognize thatthese criteria define a value and curvature (shape) for the timingsignal time-profiles that in the instant case will occur only aftersteady state has truly been reached. Therefore, one skilled in the artwill recognize that detection of steady state by these criteria does notrequire the instrument or the person using the instrument to know whenthe sample was added to the device or when the device was inserted intothe instrument, and, in particular, does not require the person toinsert the device into the instrument at any particular time.

Once all of the timing criteria discussed in the previous paragraph weremet, the instrument read the assay signals (Example 11), processed thedata and used stored calibration information to determine theconcentrations of CKMB, troponin I and Myoglobin in the blood sample.The measured concentrations were then displayed on the LCD display ofthe instrument. In order to show that the assays had reached steadystate when they were read by the instrument, the devices werere-inserted into the instrument about eleven minutes after they werefirst read (twenty six minutes after the blood was added) and read asecond time.

The results are shown in Table 15.1. The total assay time is defined asthe time between addition of the blood sample to the device and thedisplay of the measured analyte concentrations on the LCD display thefirst time the device was read. The display of the concentrationsoccurred about one minute after the instrument concluded steady statewas reached; the minute was required to scan the devices, calculateconcentrations and display the results. The second time the devices wereinserted into the meter (at twenty-six minutes), about two minuteselapsed between insertion of the devices and the display of theconcentrations. The instrument, therefore, required about one minute oftiming data acquisition to conclude that steady state had been reached.The mean and coefficient of variation (C.V.) of the CKMB, troponin I andmyoglobin concentrations measured on the six assay devices are shown inTable 15.2. The mean concentrations and the concentration C.V.s were thesame at the first (at approximately 15 minutes) and second (atapproximately 26 minutes) reading of the devices. These results showthat steady state had been reached at the time of the first reading ofthe devices.

TABLE 15.1 Measured CKMB Measured Troponin I Measured Myoglobin TotalAssay Time Concentration Concentration Concentration When the (ng/ml)(ng/ml) (ng/ml) Measurement Read at Time Read at Time Read at Time WasControlled Determined by Read At Determined by Determined by by theTiming the Timing 26 the Timing Read At 26 the Timing Read At 26 DeviceAlgorithm Algorithm minutes Algorithm minutes Algorithm minutes 1 13.86.7 6.4 1.15 1.11 98 99 2 15.3 8.5 8.5 1.35 1.31 104 108 3 15.2 6.8 6.91.30 1.21 103 103 4 15.2 7.8 7.9 1.20 1.20 111 111 5 14.7 9.1 9.3 1.551.54 110 111 6 15.5 9.2 9.2 1.34 1.26 107 109

TABLE 15.2 Time Devices Average measured concentration (ng/ml)Coefficient of Variation (%) Read CKMB Troponin I Myoglobin CKMBTroponin I Myoglobin At steady state 8.0 1.3 106 14 11 5 determined bytiming algorithm At 26 minutes 8.0 1.3 107 15 11 5

Example 16 Use of IACs to Decrease the Time to Assay Completion

This example illustrates the use of IACs to normalize an immunoassaysignal such that the normalized signal reaches steady state before theoriginal signal. The normalized signal, therefore, provides a means ofattaining assay completion and quantifying analyte concentrations morerapidly than with the unnormalized signal.

The immunoassay devices were configured with detection zones for HumanChorionic Gonadotropin (hCG), two flow controls (one with a high signaland one with a low signal) and an non-specific binding control. Humanserum spiked with 100 mIU of hCG was added to the devices. Thefluorescence from each of the detection zones was measured as a functionof time using a fluorometer of local design. The fluorometer hadessentially the same optical components as the instrument used for themeasurements discussed in example 15.

The absolute fluorescence signal from the IAC and hCG detection zones isshown in FIG. 8. The IAC and hCG assay signals all reached steady stateabout 15 minutes after the serum sample was added to the device. In mostcases, assay completion is defined to be when the assay signals reachsteady state. Therefore, in this example, the assay signal measuredafter 15 minutes would be compared with a predetermined calibrationcurve to obtain the concentration of hCG in the serum sample. The entireassay from addition of the serum sample to the device to the display ofthe hCG concentration would take slightly longer than 15 minutes.

Normalization (the method is given in the next paragraph) of the assaysignal with any of the IAC signals dramatically shortened the time forthe normalized signal to reach steady state. The normalized signal (FIG.9) reached steady state in about 8 minutes. The steady state value ofthe normalized signal was the same as the steady state value of theunnormalized signal. Therefore, the method of normalization resulted inan assay that would take only slightly longer than 8 minutes to obtainthe final concentration values and that would not change the calibrationcurve.

The assay signal was normalized to each of the IACs using equation 17.5(example 17). The function f (equation 17.4) was determined by plottingthe deviations of the assay signal at each time from the final steadystate value (equation 17.2; Ave(Assay)t=(was determined from the singledevice of this example) as a function of the deviations of the IACsignals at each time from the final steady state values (equation 17.1;Ave(IAC)t=(was determined from the single device of this example). Theresulting curves were fit with parabolic functions using the curvefitting routine of Sigma Plot (Jandel Scientific version 2.01).Therefore, the function f was a second order power series withcoefficients given by the fit.

Example 17 Equations for the Normalization of Assay Signals With One IAC

This example shows the equations used to normalize an assay signal usinga single IAC. These equations apply both to assay and IAC signals atsteady state and to assay and IAC signals that are changing with time.The concepts developed here can be extended to apply to the linearcombination of two or more IACs to normalize the assay signals tomultiple IACs that, for example, monitor both flow rate and incubationtime.

The method of normalization utilizes the deviations of the IACs and theassay signals from their respective mean steady state values. The meansteady state values are measured empirically using assay methods andassay devices identical to those in which normalization will take place.One skilled in the art will recognize that this is best accomplishedusing a variety of samples, for example, blood from about 5 to 100individuals. Additionally, diversity in samples can be achieved bypooling samples from individuals, for example, by making plasma, serumor urine pools. Diversity in the samples or sample pool used fordefining the mean of the IAC and assay signals improves the probabilitythat the normalization routine will apply to all samples to be tested.Thus, measurements should be made to obtain an accurate value for themean steady state signals. The deviations are defined as follows:Δ(IAC)_(i,t)={(IAC)_(i,t)−Ave(IAC)_(t=∞)}/Ave(IAC)_(t=∞)  Eqn. 17.1Δ(Assay)_(i,t)={(Assay)_(i,t)−Ave(Assay)_(t=∞)}/Ave(Assay)_(t=∞)  Eqn.17.2where Δ(IAC)_(i,t) and (Assay)_(i,t) are the deviations of the IAC andassay signals, respectively, for the i'th immunoassay device at time t,(IAC)_(i,t) and (Assay)_(i,t) are the IAC and assay signals,respectively, for the i'th immunoassay device at time t andAve(IAC)_(t=∞) and Ave(Assay)_(t=∞) are the mean IAC and assay signalsat steady state, respectively, determined by averaging the respectivesteady state (t=∞) signals over a sufficient number of devices. Thenumber of devices included in the average can be as low as 1 and dependson the accuracy needed and the precision of the measurement. One ofordinary skill will recognize how to determine the number of devicesrequired in the average.Algebraic rearrangement of equation 17.2 leads to the equation:Ave(Assay)_(t=∞)={(Assay)_(i,t)}/{1+Δ(Assay)_(i,t)}  Eqn. 17.3In general, the goal in any normalization scheme is to normalize theassay signal of the i'th device at any time t (i.e., (Assay)_(i,t)) toobtain the average steady state signal (i.e., Ave(Assay)_(t=∞)), sincethe average steady state signal is used to derive the calibrationinformation for the assay. The deviation Δ(Assay)_(i,t) will in generalnot be known, since the concentration of the analyte in the sample andtherefore, the expected average steady state assay signal, will not beknown. However, the deviation Δ(Assay)_(i,t) can be calculated from thedeviation Δ(IAC)_(i,t). Since the IAC signal is independent of theanalyte concentration, the expected value for the IAC signal is the sameas the average steady state value. Therefore, for each of the i assaydevices, Δ(IAC)_(i,t) can be calculated from the IAC signal,(IAC)_(i,t), and the previously determined average steady state IACsignal, Ave(IAC)_(t=∞), using equation 1. The IACs and the assays areset up such that their signals are correlated in time and/or over allthe i devices. If the IACs and assays are correlated, a relationshipwill by definition exist between their deviations, i.e., Δ(Assay)_(i,t)will be a function f of Δ(IAC)_(i,t):Δ(Assay)_(i,t) =f(Δ(IAC)_(i,t))  Eqn. 17.4Substitution of equation 17.4 into equation 17.3 and equating thenormalized assay signal norm(assay)_(i) from the i'th device withAve(Assay)_(t=∞) leads to:Norm(assay)_(i,t)={(Assay)_(i,t)}/{1+f(Δ(IAC)_(i,t))}  Eqn. 17.5Once the function f that relates the deviations of the assay and the IACis known, equation 17.5 can be used to normalize assay signals to be thesame as the mean steady state values averaged over many devices.Examples are given herein (Examples 16, 18-21) in which f is either afirst order or second order power series and in which Eqn. 17.5 (or Eqn.17.7) is used to decrease the time to assay completion (example 16), tonormalize out the effect of variable timegate times (example 19) and toimprove the accuracy and precision of the measurement of variousanalytes (examples 18, 20 and 21). One skilled in the art will recognizethat f can be many functions. In particular, f can be a Taylor seriesexpansion such that any functional relationship between the deviationsof the IAC and assay signals that obeys the criteria for defining aTaylor series can be exploited.

A special case of equation 17.4 is when the deviations of the assay andIAC signals are equal:Δ(Assay)_(i,t)=Δ(IAC)_(i,t)  Eqn. 17.6In this special case, equation 17.5 reduces to the simple ratio:Norm(assay)_(i,t)={(Assay)_(i,t)/(IAC)_(i,t)}·Ave(IAC)_(t=∞)  Eqn. 17.7

Example 18 Use of IAC to Improve Reproducibility of Analyte MeasurementAmong Plasma Samples From Several Different Individuals

Immunoassay of analytes in plasma, serum or whole blood samples can beproblematic because of the matrix effect, in which the result of theimmunoassay varies among samples from different individuals even thoughall the samples contain the same concentration of analyte. The causes ofthe matrix effect are many and depend on the exact nature of theimmunoassay. In general, the differences in the physical (viscosity,optical density, etc) and chemical (substances that interfere with theassays) properties of the samples contribute to the matrix effect. Onemethod of reducing the influence of the matrix effect is to use an IACto normalize the immunoassay. In order to work properly, the changes inthe IAC and the analyte assay associated with the matrix effect must becorrelated, i.e., the deviations of the analyte assay must be related tothe deviations of the IAC by a function (Eqn. 17.4 of example 17). Inthis example, the timegate control IAC is used to normalize and improvethe reproducibility of the measurement of CKMB on cardiac panel devices(Example 15) among plasma samples from several different individuals.

Cardiac panel devices (Example 15) were run with plasma from differentindividuals spiked with 20 ng/ml CKMB. Each sample was run on eightdevices and the results were averaged. The results from nine individualschosen at random were used to define the functional relationship betweenthe timegate control IAC and the CKMB Assay. The deviations Δ(IAC)_(i)and Δ(Assay)_(i) were calculated from equations 17.1 and 17.2respectively. The subscript t=time has been dropped because all of themeasurements were made at steady state (t=∞). The subscript i refers tothe i'th plasma sample, i.e., from the i'th individual (i=1 to 9). Thevalues of Ave(IAC) and Ave(Assay) were defined to be the mean IAC andAssay signals, respectively, averaged over the nine plasma samples.Therefore, each value, Δ(IAC)_(i) or Δ(Assay)_(i), represents thedeviation for the i'th plasma sample of the timegate control or CKMBassay signal, respectively, from the mean timegate control or CKMB assaysignal averaged over all nine plasma samples. The resultant data pairs(Δ(IAC)_(i), Δ(Assay)_(i) were plotted and fitted with a line using thecurve fitting routine of Sigma Plot (Jandel Scientific). The functionalrelationship thus obtained between the two deviations was:Δ(Assay)_(i)=0.30·Δ(IAC)_(i)  Eqn. 18.1Therefore, the normalization equation (Eqn. 17.5) is:Norm(assay)_(i)={(Assay)_(i)}/{1+0.30·Δ(IAC)_(i)}  Eqn. 18.2

To demonstrate the effect of normalization on reproducibility of analytemeasurement, plasma samples from four individuals were spiked with 20ng/ml CKMB an run on cardiac panel devices (the results from eightdevices per plasma sample were averaged). The resultant non-normalizedtimegate control signal and measured CKMB signals are shown in Table18.1. The measured CKMB signals were normalized using Eqn. 18.2. Thetimegate control signal deviation Δ(IAC)_(i) was calculated usingequation 17.1 where the value of Ave(IAC) was determined by averagingthe timegate control signals over the four plasma samples. Thenormalized CKMB signals are shown in Table 18.1. The mean non-normalizedand normalized CKMB signals averaged over the four plasma samples arethe same. However, the C.V. calculated among the four plasma samples isdramatically lower for the normalized CKMB signals. Therefore, the useof the timegate control IAC to normalize the CKMB signals dramaticallyimproved reproducibility among different plasma donors by normalizingout the matrix effects.

TABLE 18.1 Plasma Sample Timegate Control Unnormalized Normalized(Donor) Signal CKMB Signal CKMB Signal 1 157 133 119 2 105 121 123 393.8 117 123 4 93.7 106 112 Average of 1-4 112 119 119 C.V. of 1-4 27%9.4% 4.4%

Example 19 Use of an IAC to Correct an Immunoassay for Variations in theIncubation Time of the Sample With the Antibody Reagents

One potential source of imprecision or error in an immunoassay is thefailure to incubate the sample with the antibody reagents for the rightamount of time (usually until binding equilibrium is obtained). On theother hand, it may be desirable in some cases to purposely shorten theincubation time in order to shorten the time to assay completion. Ineither case, an IAC whose signal varies with the incubation time in away that is correlated with the variation in the assay signal with theincubation time can be used to normalize the assay signal. Thenormalized assay signal will be essentially independent of theincubation time of the sample with the antibody reagents. Thenormalization could, therefore, lead to improved assay precision andaccuracy and to shorter assay times.

To illustrate the use of an IAC to correct the assay signals forvariations in the time of incubation of the sample with the antibodyreagents, cardiac panel devices were constructed with timegates whosehold-time (timegate time) could be varied from 0 to 4 minutes. Humanwhole blood spiked with CKMB (5 ng/ml final), troponin I (5 ng/ml final)and myoglobin (50 ng/ml final) was run on the devices. The timegate timewas adjusted to be 0.0 minutes, 1 minute or 4 minutes. Thirty deviceswere run with each timegate time. The averaged timegate control andassay signals that were obtained for devices run with each timegate timeare shown in Table 19.1.

The timegate control and assay signals are all correlated with thetimegate time, i.e., are lower for a timegate time of 0.0 minutes thanfor the longer timegate times. The timegate control involves the bindingof an anti-morphine antibody to FETL-morphine in the reaction chamber toform a signal generating complex. The kinetics of this reaction are slowenough that the reaction is not complete when the timegate time is lessthan 1 minute. Similarly, the kinetics of the binding of the CKMB,troponin I and myoglobin to their respective antibodies on the FETLs toform a signal generating complex are slow enough that these reactionsare not complete when the timegate time is less than 1 minute. In orderto obtain the best normalization of the analyte assay signals with theIAC signals over all timegate times, the binding kinetics associatedwith forming the IAC signal generating complex should be as similar aspossible to the binding kinetics associated with forming the analyteassay signal generating complex.

The analyte assay signals in Table 19.1 were normalized with the IACsignal using the methods of example 17. The normalization equation was:Norm(assay)_(t)={(Assay)_(t)}/{1+m·Δ(IAC)_(t)}  Eqn. 19.1The subscript t refers to the timegate time in this example. Sinceindividual devices were not analyzed, the subscript i has been dropped.The deviations Δ(IAC)_(t) and Δ(assay)_(t) were determined usingequations 17.1 and 17.2, respectively, where Ave(IAC)_(t=∞) andAve(assay)_(t=∞) were calculated from the average of the values fortimegate times of 1 minute and 4 minutes. The slopes m in Eqn. 12 weredetermined by plotting the data pairs (Δ(IAC)_(t), Δ(assay)_(t)) andfitting the resultant lines using the curve fitting routine in SigmaPlot. The slopes are:

-   CKMB: m=1.0-   Troponin I: m=0.43-   Myoglobin: m=0.34    The normalized assay signals are shown in Table 19.1. The    normalization significantly corrects the signals obtained for the 0    minute timegate to be similar to the final equilibrium values    obtained for 1 minute or 4 minute timegates. Therefore, the    normalization could result in better assay precision in cases where    the imprecision in the timegate time is a factor and could also    result in faster assay times by eliminating the need for a timegate.

TABLE 19.1 Timegate Normalized Normalized Timegate Control CKMBNormalized Troponin I Troponin I Myoglobin Myoglobin Time Signal SignalCKMB Signal Signal Signal Signal Signal 0 min 49 69 108 77 91 541 616 1min 82 107 101 89 87 621 609 4 min 72 122 130 98 101 620 620 Average 6899 113 88 93 594 615 of All Times C.V.s of 25% 28% 13% 12% 7.8% 7.7%0.9% All Times

Example 20 Use of an IAC to Improve Assay Precision and Accuracy in aCyclosporin Assay

This example illustrates the use of an IAC to normalize the assaysignals from a cyclosporin immunoassay device run with whole blood toimprove the precision (C.V.) and accuracy of the assay. Three issues areaddressed: (i) the precision of the cyclosporin measurement withinsamples from single blood donors; (ii) the accuracy of the cyclosporinmeasurement in blood from four different blood donors and (iii) theaccuracy and precision of the cyclosporin measurement in blood ofdifferent hematocrit values from the same blood donor.

Whole blood from four different blood donors was spiked with cyclosporin(CS) to final total concentrations of 0 ng/ml CS, 50 ng/ml CS, 100 ng/mlCS and 800 ng/ml CS. Each spiked blood sample was run on approximately18 immunoassay devices (examples 10 and 11) with detection zones forcyclosporin and an IAC. The assay devices were inserted into aninstrument and read about 20 minutes after addition of the blood.

The integrated assay and IAC signals from each device run with the blooddonor samples spiked to 50 ng/ml CS are shown in Table 20.1 Thenormalized assay signals are also shown in the table and were calculatedfrom the assay signal and IAC signal using the following equation:normalized assay signal=(assay signal/IAC signal)×1000  Eqn. 20.1.

The C.V.s of the normalized assay signals are significantly lower thanthe C.V.s of the non-normalized assay signals.

The cyclosporin measurements at all the spiked cyclosporinconcentrations are summarized in Table 20.2. For each individual sample(i.e., each cyclosporin concentration in blood from each individualdonor), the table shows the mean and C.V. of the assay, IAC andnormalized assay signals averaged over the 18 devices run with thesample. In addition, the table shows the mean and C.V. of the assay, IACand normalized assay signals averaged over the blood samples from allfour donors (inter-donor C.V.s). The data show that the C.V. of thenormalized assay signal is less than that of the non-normalized signalfor every donor and every cyclosporin concentration tested. Furthermore,the inter-donor C.V.s of the normalized signals are significantly lowerthan those of the non-normalized signals. Therefore, the data show thatthe normalization of the assay signals with the IAC signalssignificantly improves the precision of the cyclosporin measurementwithin one sample (blood donor) and that the normalization reduces oreliminates the effect of the matrix (different blood donor samples) onthe cyclosporin measurement.

The effect of normalization on the accuracy and precision of thecyclosporin measurement was also investigated in blood samples of variedhematocrit values. The hematocrit of the blood from a single blood donorwas adjusted to three levels by centrifuging the blood and removing oradding plasma from the same donor as appropriate. Three hematocrits weretested (low=29%, medium=37% and high=45%) at three cyclosporinconcentrations: 0 ng/ml CS, 100 ng/ml CS and 400 ng/ml CS. The resultsare shown in Table 20.3. For each individual sample (i.e., eachcyclosporin concentration in blood at each hematocrit value), the tableshows the mean and C.V. of the assay, IAC and normalized assay signalsaveraged over the 18 devices run with the sample. In addition, the tableshows the mean and C.V. of the assay, IAC and normalized assay signalsaveraged over the low, medium and high hematocrit samples. The data showthat the C.V. of the normalized assay signal is less than that of thenon-normalized signal for every hematocrit and every cyclosporinconcentration tested. Furthermore, the C.V. computed by averaging thesignals from the low, medium and high hematocrit samples issignificantly lowered by normalization of the assay signal. The datatherefore show that the normalization of the assay signals with the IACsignals significantly improves the precision of the cyclosporinmeasurement within one sample (hematocrit) and that the normalizationessentially eliminates the effect of hematocrit on the cyclosporinmeasurement over the range of hematocrits tested.

In summary, all of the data presented in this example show thatnormalization of the assay signals with the IAC signals significantlyimproves the precision and accuracy of the cyclosporin measurement.

TABLE 20.1 Donor 1 Donor 2 Donor 3 Donor 4 Device Assay IAC Norm.* AssayIAC Assay IAC Norm. Assay IAC # Signal Signal Signal Signal Signal Norm.Signal Signal Signal Signal Signal Norm. 1 375 884 424 463 1048 442 385943 409 394 989 398 2 514 1080 476 431 972 443 329 819 402 514 1189 4323 423 1011 418 557 1158 481 483 1176 410 530 1211 438 4 477 1075 444 4531005 451 351 922 380 550 1240 443 5 638 1310 487 483 1042 464 495 1151430 615 1355 454 6 475 1119 425 473 1106 428 467 1145 408 631 1436 439 7521 1085 480 489 1075 455 540 1282 422 584 1303 448 8 442 936 472 344800 430 565 1238 457 540 1097 492 9 437 992 440 400 898 446 448 1034 434423 945 448 10 427 979 436 443 1012 438 409 1023 399 600 1258 477 11 414932 444 381 933 409 425 1031 412 467 1134 412 12 336 800 419 456 1018448 687 1370 501 623 1294 481 13 481 1010 476 445 1006 442 470 1147 410433 1011 428 14 497 1134 439 412 950 434 501 1187 422 608 1254 485 15345 837 412 539 1187 454 620 1378 450 468 1148 408 16 430 998 431 5031136 442 400 980 409 538 1229 438 17 406 897 453 439 1033 425 688 1290533 448 1027 436 18 506 1044 484 458 937 488 Mean 452 1007 448 454 1018446 486 1125 429 527 1183 445 C.V. 16 12 6 11 9 4 22 14 9 15 12 6*“Norm.” refers to a normalized assay signal, or a corrected assaysignal.

The cyclosporin measurements at all the spiked cyclosporinconcentrations are summarized in Table 20.2. For each individual sample(i.e., each cyclosporin concentration in blood from each individualdonor), the table shows the mean and C.V. of the assay, IAC andnormalized assay signals averaged over the 18 devices run with thesample. In addition, the table shows the mean and C.V. of the assay, IACand normalized assay signals averaged over the blood samples from allfour donors (inter-donor C.V.s). The data show that the C.V. of thenormalized assay signal is less than that of the non-normalized signalfor every donor and every cyclosporin concentration tested. Furthermore,the inter-donor C.V.s of the normalized signals are significantly lowerthan those of the non-normalized signals. Therefore, the data show thatthe normalization of the assay signals with the IAC signalssignificantly improves the precision of the cyclosporin measurementwithin one sample (blood donor) and that the normalization reduces oreliminates the effect of the matrix (different blood donor samples) onthe cyclosporin measurement.

TABLE 20.2 Norm- alized Assay IAC Assay Assay Signal IAC SignalNormalized Signal Signal CV Signal C.V. Assay Signal C.V. 0 ng/ml CSDonor 1 682 15 1113 9 610 7 Donor 2 653 18 1062 14 614 8 Donor 3 684 191154 14 . 589 7 Donor 4 753 21 1219 16 615 10 Average 693 1137 607 CV 66 2 50 ng/ml CS Donor 1 452 16 1007 12 448 6 Donor 2 454 11 1018 9 446 4Donor 3 486 22 1125 14 429 9 Donor 4 527 15 1183 12 445 6 Average 4801083 442 CV 7 8 2 100 ng/ml CS Donor 1 245 13 1012 10 242 7 Donor 2 22815 955 13 239 6 Donor 3 236 16 1083 13 218 7 Donor 4 238 17 1049 14 2276 Average 237 1025 231 CV 3 5 5 800 ng/ml CS Donor 1 101 13 1090 11 93 7Donor 2 74 14 927 15 80 8 Donor 3 82 16 1097 13 74 5 Donor 4 80 14 105414 76 5 Average 84 1042 81 CV 14 8 10

The effect of normalization on the accuracy and precision of thecyclosporin measurement was also investigated in blood samples of variedhematocrit values. The hematocrit of the blood from a single blood donorwas adjusted to three levels by centrifuging the blood and removing oradding plasma from the same donor as appropriate. Three hematocrits weretested (low=29%, medium=37% and high=45%) at three cyclosporinconcentrations: 0 ng/ml CS, 100 ng/ml CS and 400 ng/ml CS. The resultsare shown in Table 20.3. For each individual sample (i.e., eachcyclosporin concentration in blood at each hematocrit value), the tableshows the mean and C.V. of the assay, IAC and normalized assay signalsaveraged over the 18 devices run with the sample. In addition, the tableshows the mean and C.V. of the assay, IAC and normalized assay signalsaveraged over the low, medium and high hematocrit samples. The data showthat the C.V. of the normalized assay signal is less than that of thenon-normalized signal for every hematocrit and every cyclosporinconcentration tested. Furthermore, the C.V. computed by averaging thesignals from the low, medium and high hematocrit samples issignificantly lowered by normalization of the assay signal. The datatherefore show that the normalization of the assay signals with the IACsignals significantly improves the precision of the cyclosporinmeasurement within one sample (hematocrit) and that the normalizationessentially eliminates the effect of hematocrit on the cyclosporinmeasurement over the range of hematocrits tested.

In summary, all of the data presented in this example show thatnormalization of the assay signals with the IAC signals significantlyimproves the precision and accuracy of the cyclosporin measurement.

TABLE 20.3 Norm- Assay IAC alized Normalized Assay Signal IAC SignalAssay Assay Signal CV Signal CV Signal Signal C.V. 0 ng/ml CS Low 421 10509 11 829 3 Med 520 12 645 10 806 5 High 741 10 848 10 877 7 Average561 667 837 CV 29 26 4 100 ng/ml CS Low 235 9 507 9 464 6 Med 289 11 6379 453 4 High 342 13 705 11 486 6 Average 288 616 468 CV 19 16 4 400ng/ml CS Low 93 10 457 10 203 6 Med 103 8 565 12 184 8 High 143 14 80011 179 12 Average 113 607 189 CV 23 29 7

Example 21 Use of an IAC to Improve the Precision of an Assay for CKMB,Troponin I and Myoglobin

Human plasma spiked with CKMB (5 ng/ml), troponin I (2 ng/ml) andmyoglobin (70 ng/ml) was assayed for these analytes on cardiac panelimmunoassay devices (23 devices) which were read in an instrument asdescribed in previous examples. The assay signals for CKMB, troponin Iand myoglobin measured in each immunoassay device are shown in Table21.1. The normalized assay signals are also shown in the table and werecalculated from the flow control signal and the assay signals usingequation 17.7 (example 17). The C.V.s of the normalized signals aresignificantly lower than those of the non-normalized signals. Therefore,the data show that normalization of the assay signals with the IAC flowcontrol improves the precision the CKMB, troponin I and myoglobinassays.

TABLE 21.1 CKMB Troponin I Myoglobin Flow Control Assay Normalized AssayNormalized Assay Normalized Device # Signal Signal Assay Signal SignalAssay Signal Signal Assay Signal  1 70.9 14.3 13.6 15.2 14.5 94.0 89.4 2 75.2 13.9 12.5 15.7 14.0 97.4 87.2  3 71.7 14.4 13.6 14.8 13.9 97.291.4  4 63.1 13.0 13.8 11.4 12.2 88.2 94.3  5 62.0 11.4 12.3 13.9 15.280.1 87.1  6 71.1 12.7 12.0 14.2 13.4 82.9 78.7  7 65.1 11.9 12.3 12.813.3 82.4 85.2  8 79.1 15.8 13.5 15.0 12.7 95.3 81.2  9 65.8 12.4 12.713.7 14.1 95.9 98.2 10 62.9 12.9 13.8 14.8 15.9 86.2 92.3 11 67.5 15.715.7 14.2 14.2 96.2 96.0 12 59.4 10.4 11.8 11.0 12.4 75.6 85.9 13 67.69.8 9.8 11.4 11.4 80.8 80.5 14 69.4 12.0 11.6 14.5 14.1 89.2 86.6 1572.8 14.8 13.7 15.3 14.1 104.2 96.5 16 71.0 13.3 12.6 14.4 13.7 91.686.9 17 61.4 11.4 12.5 12.0 13.2 79.1 86.9 18 59.5 9.6 10.9 11.2 12.670.4 79.7 19 64.7 12.2 12.7 13.6 14.2 88.5 92.3 20 73.3 13.7 12.6 13.412.3 91.4 84.0 21 64.3 11.4 11.9 12.3 12.9 84.1 88.2 22 64.3 12.1 12.710.8 11.3 72.6 76.1 23 68.3 10.6 10.5 12.9 12.8 78.7 77.6 Average 67.412.6 12.6 13.4 13.4 87.0 87.1 C.V. 7.7 13.7 9.9 11.4 8.3 10.1 7.1

The various embodiments of the invention described above may beimplemented using hardware, software or a combination thereof and may beimplemented in a computer system or other processing system. In fact, inone embodiment, these elements are implemented using a computer systemcapable of carrying out the functionality described with respectthereto. An example computer system 702 is shown in FIG. 18. Thecomputer system 702 includes one or more processors, such as processor704. The processor 704 is connected to a communication bus 706. Varioussoftware embodiments are described in terms of this example computersystem. After reading this description, it will become apparent to aperson skilled in the relevant art how to implement the invention usingother computer systems and/or computer architectures.

Computer system 702 also includes a main memory 708, preferably randomaccess memory (RAM), and can also include a secondary memory 710. Thesecondary memory 710 can include, for example, a hard disk drive 712and/or a removable storage drive 714, representing a floppy disk drive,a magnetic tape drive, an optical disk drive, etc. The removable storagedrive 714 reads from and/or writes to a removable storage medium 718 ina well known manner. Removable storage media 718, represents a floppydisk, magnetic tape, optical disk, etc. which is read by and written toby removable storage drive 714. As will be appreciated, the removablestorage media 718 includes a computer usable storage medium havingstored therein computer software and/or data.

In alternative embodiments, secondary memory 710 may include othersimilar means for allowing computer programs or other instructions to beloaded into computer system 702. Such means can include, for example, aremovable storage unit 722 and an interface 720. Examples of such caninclude a program cartridge and cartridge interface (such as that foundin video game devices), a removable memory chip (such as an EPROM, orPROM) and associated socket, and other removable storage units 722 andinterfaces 720 which allow software and data to be transferred from theremovable storage unit 718 to computer system 702.

Computer system 702 can also include a communications interface 724.Communications interface 724 allows software and data to be transferredbetween computer system 702 and external devices. Examples ofcommunications interface 724 can include a modem, a network interface(such as an Ethernet card), a communications port, a PCMCIA slot andcard, etc. Software and data transferred via communications interface724 are in the form of signals which can be electronic, electromagnetic,optical or other signals capable of being received by communicationsinterface 724. These signals are provided to communications interfacevia a channel 728. This channel 728 carries signals and can beimplemented using a wireless medium, wire or cable, fiber optics, orother communications medium. Some examples of a channel can include aphone line, a cellular phone link, an RF link, a network interface, andother communications channels.

In this document, the terms “computer program medium” and “computerusable medium” are used to generally refer to media such as removablestorage device 718, a hard disk installed in hard disk drive 712, andsignals on channel 728. These computer program products are means forproviding software to computer system 702.

Computer programs (also called computer control logic) are stored inmain memory and/or secondary memory 710. Computer programs can also bereceived via communications interface 724. Such computer programs, whenexecuted, enable the computer system 702 to perform the features of thepresent invention as discussed herein. In particular, the computerprograms, when executed, enable the processor 704 to perform thefeatures of the present invention. Accordingly, such computer programsrepresent controllers of the computer system 702.

In an embodiment where the elements are implemented using software, thesoftware may be stored in a computer program product and loaded intocomputer system 702 using removable storage drive 714, hard drive 712 orcommunications interface 724. The control logic (software), whenexecuted by the processor 704, causes the processor 704 to perform thefunctions of the invention as described herein.

In another embodiment, the elements are implemented primarily inhardware using, for example, hardware components such as applicationspecific integrated circuits (ASICs). Implementation of the hardwarestate machine so as to perform the functions described herein will beapparent to persons skilled in the relevant art(s). In yet anotherembodiment, elements are implemented using a combination of bothhardware and software.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The methods, apparatus,computer programmable media, assay devices, and kits of the inventionare representative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. Modificationstherein and other uses will occur to those skilled in the art. Thesemodifications are encompassed within the spirit of the invention and aredefined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are set forth within the following claims.

1. An apparatus for measuring progress and time of completion of anassay in an assay device, comprising: (a) said assay device comprising areaction chamber and at least one diagnostic lane, wherein a label isprovided in said reaction chamber; (b) an optical component fordetecting a signal generated from said label in at least one discretezone of said diagnostic lane; and (c) a signal processor for determiningsaid progress and time of completion of said assay in said assay devicefrom at least one parameter selected from the group consisting of a rateof change of the amount of said signal and an absolute amount of saidsignal; wherein the label does not appreciably bind to assay reagents insaid assay device.
 2. A kit for measuring progress and time ofcompletion of an assay in an assay device, comprising: (a) instructionscomprising text or diagrams about the apparatus and/or kit; and (b) anapparatus, comprising: (i) said assay device comprising a reactionchamber and at least one diagnostic lane, wherein a label is provided insaid reaction chamber; (ii) an optical component for detecting a signalgenerated from said label in at least one discrete zone of saiddiagnostic lane; and (iii) a signal processor for determining saidprogress and time of completion of said assay in said assay device fromat least one parameter selected from the group consisting of a rate ofchange of the amount of said signal and an absolute amount of saidsignal; wherein the label does not appreciably bind to assay reagents insaid assay device.
 3. An apparatus for measuring progress and time ofcompletion of an assay in an assay device, comprising: (a) said assaydevice comprising a reaction chamber, and at least one diagnostic lane,wherein said diagnostic lane is in fluid communication with saidreaction chamber when fluid and a detectable label are added to saidreaction chamber; (b) an optical component for detecting a signalgenerated from said label in at least one discrete zone of saiddiagnostic lane; and (c) a signal processor for determining saidprogress and time of completion of said assay in said assay device fromat least one parameter selected from the group consisting of a rate ofchange of the amount of said signal and an absolute amount of saidsignal; wherein the label does not appreciably bind to assay reagents insaid assay device.
 4. The apparatus of claim 1, wherein said label islinked to a member of a binding pair.
 5. The apparatus of claim 4,wherein the member of a binding pair is selected from the groupconsisting of binding protein, antibody, antibody fragment, protein,peptide, and organic molecule.
 6. The apparatus of claim 1, wherein saidassay reagents are selected from the group consisting of bindingprotein, antibody, antibody fragment, protein, peptide, and organicmolecule.
 7. The apparatus of claim 1, wherein said label is selectedfrom the group of molecules consisting of dye, fluorescence emittingdye, chemiluminescence emitting dye, infrared emitting dye, colloidalsol, molecule that generates an electrical and/or magnetic signal, andenzyme.
 8. The kit of claim 2, wherein said label is linked to a memberof a binding pair.
 9. The kit of claim 8, wherein the member of abinding pair is selected from the group consisting of binding protein,antibody, antibody fragment, protein, peptide, and organic molecule. 10.The kit of claim 2, wherein said label is selected from the group ofmolecules consisting of dye, fluorescence emitting dye,chemiluminescence emitting dye, infrared emitting dye, colloidal sol,molecule that generates an electrical and/or magnetic signal, andenzyme.
 11. The kit of claim 2, wherein said assay reagents are selectedfrom the group consisting of binding protein, antibody, antibodyfragment, protein, peptide, and organic molecule.
 12. The apparatus ofclaim 3, wherein said label is linked to a member of a binding pair. 13.The apparatus of claim 12, wherein the member of a binding pair isselected from the group consisting of binding protein, antibody,antibody fragment, protein, peptide, and organic molecule.
 14. Theapparatus of claim 3, wherein said label is selected from the group ofmolecules consisting of dye, fluorescence emitting dye,chemiluminescence emitting dye, infrared emitting dye, colloidal sol,molecule that generates an electrical and/or magnetic signal, andenzyme.
 15. The apparatus of claim 3, wherein said assay reagents areselected from the group consisting of binding protein, antibody,antibody fragment, protein, peptide, and organic molecule.